This review is written with the goal of informing public health concerns related to nanoscience, while raising awareness of nanomaterials toxicity among scientists and manufacturers handling them. We show that humans have always been exposed to nanoparticles and dust from natural sources and human activities, the recent development of industry and combustion-based engine transportation profoundly increasing anthropogenic nanoparticulate pollution. The key to understanding the toxicity of nanoparticles is that their minute size, smaller than cells and cellular organelles, allows them to penetrate these basic biological structures, disrupting their normal function. Among diseases associated with nanoparticles are asthma, bronchitis, lung cancer, neurodegenerative diseases (such as Parkinson`s and Alzheimer`s diseases), Crohn`s disease, colon cancer. Nanoparticles that enter the circulatory system are related to occurrence of arteriosclerosis, and blood clots, arrhythmia, heart diseases, and ultimately cardiac death. We show that possible adverse effects of nanoparticles on human health depend on individual factors such as genetics and existing disease, as well as exposure, and nanoparticle chemistry, size, shape, and agglomeration state. The faster we will understand their causes and mechanisms, the more likely we are to find cures for diseases associated with nanoparticle exposure. We foresee a future with better-informed, and hopefully more cautious manipulation of engineered nanomaterials, as well as the development of laws and policies for safely managing all aspects of nanomaterial manufacturing, industrial and commercial use, and recycling.

Nanomaterials and nanoparticles: Sources and toxicity

This review is presented as a common foundation for scientists interested in nanoparticles, their origin, activity, and biological toxicity. It is written with the goal of rationalizing and informing public health concerns related to this sometimes-strange new science of “nano,” while raising awareness of nanomaterials’ toxicity among scientists and manufacturers handling them. We show that humans have always been exposed to tiny particles via dust storms, volcanic ash, and other natural processes, and that our bodily systems are well adapted to protect us from these potentially harmful intruders. The reticuloendothelial system, in particular, actively neutralizes and eliminates foreign matter in the body, including viruses and nonbiological particles. Particles originating from human activities have existed for millennia, e.g., smoke from combustion and lint from garments, but the recent development of industry and combustion-based engine transportation has profoundly increased anthropogenic particulate pollution. Significantly, technological advancement has also changed the character of particulate pollution, increasing the proportion of nanometer-sized particles-“nanoparticles”-and expanding the variety of chemical compositions. Recent epidemiological studies have shown a strong correlation between particulate air pollution levels, respiratory and cardiovascular diseases, various cancers, and mortality. Adverse effects of nanoparticles on human health depend on individual factors such as genetics and existing disease, as well as exposure, and nanoparticle chemistry, size, shape, agglomeration state, and electromagnetic properties. Animal and human studies show that inhaled nanoparticles are less efficiently removed than larger particles by the macrophage clearance mechanisms in the lungs, causing lung damage, and that nanoparticles can translocate through the circulatory, lymphatic, and nervous systems to many tissues and organs, including the brain. The key to understanding the toxicity of nanoparticles is that their minute size, smaller than cells and cellular organelles, allows them to penetrate these basic biological structures, disrupting their normal function. Examples of toxic effects include tissue inflammation, and altered cellular redox balance toward oxidation, causing abnormal function or cell death. The manipulation of matter at the scale of atoms, “nanotechnology,” is creating many new materials with characteristics not always easily predicted from current knowledge. Within the nearly limitless diversity of these materials, some happen to be toxic to biological systems, others are relatively benign, while others confer health benefits. Some of these materials have desirable characteristics for industrial applications, as nanostructured materials often exhibit beneficial properties, from UV absorbance in sunscreen to oil-less lubrication of motors. A rational science-based approach is needed to minimize harm caused by these materials, while supporting continued study and appropriate industrial development. As current knowledge of the toxicology of “bulk” materials may not suffice in reliably predicting toxic forms of nanoparticles, ongoing and expanded study of “nanotoxicity” will be necessary. For nanotechnologies with clearly associated health risks, intelligent design of materials and devices is needed to derive the benefits of these new technologies while limiting adverse health impacts. Human exposure to toxic nanoparticles can be reduced through identifying creation-exposure pathways of toxins, a study that may someday soon unravel the mysteries of diseases such as Parkinson’s and Alzheimer’s. Reduction in fossil fuel combustion would have a large impact on global human exposure to nanoparticles, as would limiting deforestation and desertification. While nanotoxicity is a relatively new concept to science, this review reveals the result of life’s long history of evolution in the presence of nanoparticles, and how the human body, in particular, has adapted to defend itself against nanoparticulate intruders.

https://avs.scitation.org/doi/pdf/10.1116/1.2815690

Biologically formed nanoparticles of the strongly magnetic mineral, magnetite, were first detected in the human brain over 20 y ago [Kirschvink JL, Kobayashi-Kirschvink A, Woodford BJ (1992) Proc Natl Acad Sci USA 89(16):7683-7687]. Magnetite can have potentially large impacts on the brain due to its unique combination of redox activity, surface charge, and strongly magnetic behavior. We used magnetic analyses and electron microscopy to identify the abundant presence in the brain of magnetite nanoparticles that are consistent with high-temperature formation, suggesting, therefore, an external, not internal, source. Comprising a separate nanoparticle population from the euhedral particles ascribed to endogenous sources, these brain magnetites are often found with other transition metal nanoparticles, and they display rounded crystal morphologies and fused surface textures, reflecting crystallization upon cooling from an initially heated, iron-bearing source material. Such high-temperature magnetite nanospheres are ubiquitous and abundant in airborne particulate matter pollution. They arise as combustion-derived, iron-rich particles, often associated with other transition metal particles, which condense and/or oxidize upon airborne release. Those magnetite pollutant particles which are <∼200 nm in diameter can enter the brain directly via the olfactory bulb. Their presence proves that externally sourced iron-bearing nanoparticles, rather than their soluble compounds, can be transported directly into the brain, where they may pose hazard to human health.

https://www.pnas.org/content/pnas/113/39/10797.full.pdf

Magnetite pollution nanoparticles in the human brain.

Metals in Alzheimer's and Parkinson's Diseases

Approximately a third of the human proteome contains metal cations, either in form of cofactors with catalytic functions, or as structural support elements.1,2 To guarantee a proper maintenance of this metal ion pool, both at the cellular and whole organism levels, nature has evolved a highly sophisticated machinery comprised of a complex interplay between DNA, proteins, and biomolecules.3 Over the past decades, a steadily growing number of diseases have been identified, which are characterized by metal imbalance in cells and tissues. Among the most prominent examples rank Alzheimer’s disease and Parkinson’s disease, two neurodegenerative disorders that involve abnormal accumulation of transition metals in brain tissue.4 While some progress has been made at understanding the molecular basis of these disorders, many important questions remain unanswered. For example, little is known about the cellular structures that are involved in transiently storing metal ions prior to their incorporation into metalloproteins or the fate of metal ions upon protein degradation. An important first step towards unraveling the regulatory mechanisms involved in trace metal transport, storage, and distribution represents the identification and quantification of the metals, ideally in context of their native physiological environment in tissues, cells, or even at the level of individual organelles and subcellular compartments.

Since the inception of the first histochemical methods for the microscopic demonstration of transition metals in tissues more than 140 years ago,5 many highly sensitive microanalytical techniques and instruments have been developed for the in situ analysis of trace metals. The aim of this review is to provide an overview of the most recent achievements in trace metal imaging while at the same time also offering a historical perspective of this rapidly evolving research field. Although this survey has been structured according to the various analytical techniques, particular emphasis is given to the biological background for a better understanding of the context and importance of each discussed study.

An overview of the most important microanalytical techniques currently available for the in situ detection of trace metals in cells and tissues is compiled in Table 1. Depending on the task, each technique may offer specific advantages and, of course, also disadvantages. Currently, synchrotron- and focused ion-beam microprobes presumably offer the best combination of sensitivity and spatial resolution; however, the ionizing high-energy excitation beam is not compatible with studying live organisms. Conversely, techniques that have been specifically developed for physiological imaging in clinical medicine, notably magnetic resonance imaging and positron emission tomography, inherently offer only a low spatial resolution and are merely suitable for obtaining information at the organ or tissue level. Although fluorescence microscopy based methods provide very high sensitivity down to the single molecule level while being at the same time compatible with live cell and tissue studies, scattering and limited penetration depth renders these techniques unsuitable for imaging opaque specimens. There are also important differences regarding the type of quantitative information that can be gained by each of these analytical techniques. For example, the histochemical detection with chromogenic and fluorogenic dyes relies on a competitive exchange of the metal ion within its native environment, most likely coordinated to endogenous ligands. Depending on the exchange kinetics and thermodynamic affinity of the histochemical indicator, only a fraction of the total metal ion contents in a cell or tissue can be probed. Nevertheless, this kinetically labile pool is particularly of interest in context of understanding the uptake, distribution, and regulation of trace elements at the cellular level, and in this regard, these methods offer unique opportunities to dynamically image metal ion fluxes in live cells with high sensitivity and spatial resolution. At the same time, organelles and proteins of interest can be readily labeled with genetically encoded green fluorescent protein tags,12 thus providing direct insights into dynamic processes within a larger cellular and biochemical context. In contrast, similar correlative information is difficult to gain with the fully quantitative micro beam methods, which require xenobiotic elemental tags for identifying subcellular structures. Autoradiographic tracer experiments offer much improved resolution over PET; however, the technique is only applicable to fixed or frozen tissues and cells. Furthermore, tracer studies cannot provide direct information regarding the endogenous metal composition of cells or tissues, and are therefore primarily limited to metal uptake, distribution, and release studies. Finally, mass spectrometric analyses are surface-based methods that destroy the sample while measuring its elemental composition. Clearly, only the combination of several analytical techniques and specific biochemical studies may lead to a fully comprehensive analysis of a biological system.



Table 1

Spatially resolved microanalytical techniques for in situ imaging of trace metals in biology.6–11




2. Histochemical Techniques
Histology is the branch of biology dealing with the study of microscopic anatomy of cells and tissues of plants and animals. Histological studies are typically carried out on thin sections of tissue or with cultured cells. To visualize and identify particular structures, a broad spectrum of histological stains and indicators are available. Among the most widely used dyes are hematoxylin and eosin, which stain nuclei blue and the cytoplasm pink, respectively.13 The history of detecting biological trace metal by histological methods dates back more than 140 years. Although these techniques have been today mostly replaced by the much more sensitive modern analytical methods described in this review article, histochemical approaches for visualizing metals mark the very beginning in the exploration of the inorganic physiology of transition metals. Given this special place in history, we deemed it necessary to briefly review some of the early achievements in this field.

2.1. Chromogenic Detection with Chelators and Ligands
Ever since the inception of Perls Prussian blue method for staining of non-heme iron, numerous indicators have been developed for the in situ visualization of trace metals in biological tissues and cells.13 Due to their limited sensitivity; however, most of these techniques were only suitable for the diagnosis of pathological conditions, typically associated with excess metal accumulations, thus preventing their application for routine staining of normal tissue. Furthermore, because the dyes are engaged in a competitive exchange equilibrium with endogenous ligands, histological stains are not suitable for the analytical determination of the total metal contents in tissues and thus limited to the visualization of the histologically reactive fraction of loosely bound labile metal ions.



2.1.1. Histochemistry of Iron 
The histochemical demonstration of labile iron reported by Perls in 1867 is among the earliest accounts describing the in situ visualization of a trace metal in biological tissues.5 The method was originally described by Grohe, who observed the formation of a blue coloration when he treated cadaver tissues with potassium ferrocyanide in acidic solution.14 Due to its low cost and simplicity, the technique is still used today for the histological visualization of non-heme iron. Some variations focused on optimizing the concentrations and proportions of the reagents,15–17 among which Lison’s protocol17 appears to be most popular today. An intensification of Perls’ staining can be obtained by exploiting the use of ferric ferrocyanide in catalyzing the oxidation of diaminobenzidine (DAB) to polymeric benzidine black by hydrogen peroxide.18

An alternative method employs the reaction of ferricyanide with Fe(II) resulting in Turnbull blue.19 Since almost all of the Fe in tissues is in the ferric form, the staining procedure requires the in situ conversion of Fe(III) to Fe(II) with ammonium sulfide.15 Due to often incomplete reduction, the method never gained much attention. More recently, an application of Turnbull blue, named the ‘perfusion Turnbull method’ has been developed, where in vivo perfusion of acidic ferricyanide is followed by DAB intensification.20 The direct in vivo perfusion avoids artifacts associated with tissue fixation, including the loss of loosely bound iron and oxidation of Fe(II) to Fe(III). Similarly, Perls method was modified by employing in vivo perfusion with acidic ferrocyanide. Both methods are capable of identifying organs and tissues containing histochemically reactive iron over a broad pH range, including the low endosomal pH.21,22

The history of iron histochemistry would be incomplete without mentioning Quincke’s method, which employed ammonium sulfide for the precipitation of tissue iron as its sulfide.23 A detailed account on the various techniques, including a comprehensive historical overview of non-heme iron chemistry, has been recently published.24

I. Ferrer

Papers

Study of the localization of iron, ferritin, and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level.