Ali Kermanizadeh

An in vitro assessment of panel of engineered nanomaterials using a human renal cell line: cytotoxicity, pro-inflammatory response, oxidative stress and genotoxicity

BackgroundIt has been shown that nanomaterials (NMs) are able to translocate to secondary tissues one of the important being the kidneys. Oxidative stress has been implicated as a possible mechanism for NM toxicity, hence effects on the human renal proximal tubule epithelial cells (HK-2) treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO - coated - NM 110 and uncoated - NM 111), two multi walled carbon nanotubes (MWCNT) (NM 400 and NM 402), one silver (NM 300) and five TiO2 NMs (NM 101, NRCWE 001, 002, 003 and 004) were evaluated.MethodsIn order to assess the toxicological impact of the engineered NMs on HK-2 cells - WST-1 cytotoxicity assay, FACSArray, HE oxidation and the comet assays were utilised. For statistical analysis, the experimental values were compared to their corresponding controls using an ANOVA with Tukey’s multiple comparison.ResultsWe found the two ZnO NMs (24 hr LC50 – 2.5 μg/cm2) and silver NM (24 hr LC50 – 10 μg/cm2) were highly cytotoxic to the cells. The LC50 was not attained in the presence of any of the other engineered nanomaterials (up to 80 μg/cm2). All nanomaterials significantly increased IL8 and IL6 production. Meanwhile no significant change in TNF-α or MCP-1 was detectable. The most notable increase in ROS was noted following treatment with the Ag and the two ZnO NMs. Finally, genotoxicity was measured at sub-lethal concentrations. We found a small but significant increase in DNA damage following exposure to seven of the ten NMs investigated (NM 111, NRCWE 001 and NRCWE 003 being the exception) with this increase being most visible following exposure to Ag and the positively charged TiO2.ConclusionsWhile the NMs could be categorised as low and highly cytotoxic, sub-lethal effects such as cytokine production and genotoxicity were observed with some of the low toxicity materials.

In vitro assessment of engineered nanomaterials using a hepatocyte cell line: cytotoxicity, pro-inflammatory cytokines and functional markers

Abstract Effects on the liver C3A cell line treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO; coated 100 nm and uncoated 130 nm), two multi-walled carbon nanotubes (MWCNTs), one silver (Ag < 20 nm), one 7 nm anatase, two rutile TiO2 nanoparticles (10 and 94 nm) and two derivatives with positive and negative covalent functionalisation of the 10 nm rutile were evaluated. The silver particles elicited the greatest level of cytotoxicity (24 h LC50 – 2 µg/cm2). The silver was followed by the uncoated ZnO (24 h LC50 – 7.5 µg/cm2) and coated ZnO (24 h LC50 – 15 µg/cm2) particles with respect to cytotoxicity. The ZnO NMs were found to be about 50–60% soluble which could account for their toxicity. By contrast, the Ag was <1% soluble. The LC50 was not attained in the presence of any of the other engineered NMs (up to 80 µg/cm2). All NMs significantly increased IL-8 production. Meanwhile, no significant change in TNF-α, IL-6 or CRP was detected. Urea and albumin production were measured as indicators of hepatic function. These markers were only altered by the coated and uncoated ZnO, which significantly decreased albumin production.

Effects of Silver Nanoparticles on the Liver and Hepatocytes in vitro

With the increasing use and incorporation of nanoparticles (NPs) into consumer products, screening for potential toxicity is necessary to ensure customer safety. NPs have been shown to translocate to the bloodstream following inhalation and ingestion, and such studies demonstrate that the liver is an important organ for accumulation. Silver (Ag) NPs are highly relevant for human exposure due to their use in food contact materials, dietary supplements, and antibacterial wound treatments. Due to the large number of different NPs already used in various products and being developed for new applications, it is essential that relevant, quick, and cheap methods of in vitro risk assessment suitable for these new materials are established. Therefore, this study used a simple hepatocytes model combined with an in vivo injection model to simulate the passage of a small amount of NPs into the bloodstream following exposure, e.g., via ingestion or inhalation, and examined the potential of Ag NPs of 20 nm diameter to cause toxicity, inflammation, and oxidative stress in the liver following in vivo exposures of female Wistar rats via iv injection to 50 μg of NPs and in vitro exposures using the human hepatocyte cell line C3A. We found that Ag NPs were highly cytotoxic to hepatocytes (LC(50) lactate dehydrogenase: 2.5 μg/cm(2)) and affected hepatocyte homeostasis by reducing albumin release. At sublethal concentrations with normal cell or tissue morphology, Ag NPs were detected in cytoplasm and nuclei of hepatocytes. We observed similar effects of Ag NPs on inflammatory mediator expression in vitro and in vivo with increase of interleukin-8 (IL-8)/macrophage inflammatory protein 2, IL-1RI, and tumor necrosis factor-α expression in both models and increased IL-8 protein release in vitro. This article presents evidence of the potential toxicity and inflammogenic potential of Ag NPs in the liver following ingestion. In addition, the similarities between in vitro and in vivo responses are striking and encouraging for future reduction, refinement, and replacement of animal studies by the use of hepatocyte cell lines in particle risk assessment.

An in vitro liver model - assessing oxidative stress and genotoxicity following exposure of hepatocytes to a panel of engineered nanomaterials

BackgroundFollowing exposure via inhalation, intratracheal instillation or ingestion some nanomaterials (NM) have been shown to translocate to the liver. Since oxidative stress has been implicated as a possible mechanism for NM toxicity this study aimed to investigate the effects of various materials (five titanium dioxide (TiO2), two zinc oxide (ZnO), two multi-walled carbon nanotubes (MWCNT) and one silver (Ag) NM) on oxidative responses of C3A cell line as a model for potential detrimental properties of nanomaterials on the liver.ResultsWe noted a dose dependant decrease in the cellular glutathione content following exposure of the C3A cells to Ag, the ZnO and the MWCNTs. Intracellular ROS levels were also measured and shown to increase significantly following exposure of the C3A to the low toxicity NMs (MWCNT and TiO2). The antioxidant Trolox in part prevented the detrimental effect of NMs on cell viability, and decreased the NM induced IL8 production after exposure to all but the Ag particulate. Following 4 hr exposure of the C3A cells to sub-lethal levels of the NMs, the largest amount of DNA damage was induced by two of the TiO2 samples (7 nm and the positively charged 10 nm particles).ConclusionsAll ten NMs exhibited effects on the hepatocyte cell line that were at least in part ROS/oxidative stress mediated. These effects included mild genotoxicity and IL8 production for all NM except the Ag possibly due to its highly cytotoxic nature.

A Multilaboratory Toxicological Assessment of a Panel of 10 Engineered Nanomaterials to Human Health-ENPRA Project-The Highlights, Limitations, and Current and Future Challenges

ABSTRACT ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of “nanotoxicology research” based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

Role of oxidative stress in carbon nanotube-generated health effects

Abstract The development of products containing carbon nanotubes (CNTs) is a major achievement of nanotechnology, although concerns regarding risk of toxic effects linger if the hazards associated with these materials are not thoroughly investigated. Exposure to CNTs has been associated with depletion of antioxidants, increased intracellular production of reactive oxygen species and pro-inflammatory signaling in cultured cells with primary function in the immune system as well as epithelial, endothelial and stromal cells. Pre-treatment with antioxidants has been shown to attenuate these effects, indicating a dependency of oxidative stress on cellular responses to CNT exposure. CNT-mediated oxidative stress in cell cultures has been associated with elevated levels of lipid peroxidation products and oxidatively damaged DNA. Investigations of oxidative stress endpoints in animal studies have utilized pulmonary, gastrointestinal, intravenous and intraperitoneal exposure routes, documenting elevated levels of lipid peroxidation products and oxidatively damaged DNA nucleobases especially in the lungs and liver, which to some extent occur concomitantly with altered levels of components in the antioxidant defense system (glutathione, superoxide dismutase or catalase). CNTs are biopersistent high aspect ratio materials, and some are rigid with lengths that lead to frustrated phagocytosis and pleural accumulation. There is accumulating evidence showing that pulmonary exposure to CNTs is associated with fibrosis and neoplastic changes in the lungs, and cardiovascular disease. As oxidative stress and inflammation responses are implicated in the development of these diseases, converging lines of evidence indicate that exposure to CNTs is associated with increased risk of cardiopulmonary diseases through generation of a pro-inflammatory and pro-oxidant milieu in the lungs.

Investigating the relationship between nanomaterial hazard and physicochemical properties: Informing the exploitation of nanomaterials within therapeutic and diagnostic applications

Nanomaterials (NMs) have the potential to improve the treatment and diagnosis of disease as they are suitable candidates for a number of diagnostic and therapeutic applications. On entering the body via a variety of exposure routes, and during their translocation to secondary target sites it is inevitable that NMs interact with biological molecules, such as proteins. These interactions may influence the behaviour and toxicity of NMs following exposure. As the surface of NMs is what interacts with cells and tissues it is necessary to identify the influence of NM surface properties on their toxicity, and determine how this is influenced by the route of exposure, and physico-chemical characteristics of NMs. The term protein corona is used to describe the coating of the NM surface with protein. The protein corona is a dynamic and complex structure whose composition is dictated by the biological medium and the physico-chemical properties of NMs (such as their size, composition, hydrophobicity and charge) as this influences protein binding specificity and affinity. Depending on the route of exposure (e.g. inhalation or injection) NMs will encounter different proteins. We have observed that i) the composition of protein corona of NMs is likely to be dictated by their route of entry, ii) the translocation of NMs to secondary target sites may influence the composition of the protein corona (i.e. they encounter different proteins on their transport in the body) so that the composition of the protein corona evolves over time, iii) the physico-chemical characteristics of NMs dictate the composition of the protein corona, and the toxicity of NMs and iv) NMs can affect secondary target sites that vary according to delivery route and corona composition following exposure. These findings, and evidence from the wider literature has therefore led us to hypothesise that NM toxicity is dictated by the exposure route due to the acquisition of a surface coating (protein corona) that is determined by the route of entry and physico-chemical properties of the NM. This information can be exploited within the intelligent design of NMs in the future (e.g. to control protein adsorption and the subsequent cellular response), and be used to improve the design of toxicology investigations (e.g. to inform how NMs should be dispersed within in vitro experiments to more accurately reflect in vivo conditions).

Nanomaterial translocation–the biokinetics, tissue accumulation, toxicity and fate of materials in secondary organs–a review

Engineered nanomaterials (NMs) offer great technological advantages but their risks to human health are still not fully understood. An increasing body of evidence suggests that some NMs are capable of distributing from the site of exposure to a number of secondary organs. The research into the toxicity posed by the NMs in these secondary organs is expanding due to the realisation that some materials may reach and accumulate in these target sites. The translocation to secondary organs includes, but is not limited to, the hepatic, central nervous, cardiovascular and renal systems. Current data indicates that pulmonary exposure is associated with low (inhalation route–0.00001–1% of total applied dose–24 h) translocation of virtually insoluble NMs such as iridium, carbon black, gold and polystyrene, while slightly higher translocation has been observed for NMs with either slow (e.g. silver, cerium dioxide and quantum dots) or fast (e.g. zinc oxide) solubility. The translocation of NMs following intratracheal, intranasal and pharyngeal aspiration is higher (up to 10% of administered dose), however the relevance of these routes for risk assessment is questionable. Uptake of the materials from the gastrointestinal tract seems to follow the same pattern as inhalation translocation, whereas the dermal uptake of NMs is generally reported to be low. The toxicological effects in secondary organs include oxidative stress, inflammation, cytotoxicity and dysfunction of cellular and physiological processes. For toxicological and risk evaluation, further information on the toxicokinetics and persistence of NMs is crucial. The overall aim of this review is to outline the data currently available in the literature on the biokinetics, accumulation, toxicity and eventual fate of NMs in order to assess the potential risks posed by NMs to secondary organs.

Atherosclerosis and vasomotor dysfunction in arteries of animals after exposure to combustion-derived particulate matter or nanomaterials

Abstract Exposure to particulate matter (PM) from traffic vehicles is hazardous to the vascular system, leading to clinical manifestations and mortality due to ischemic heart disease. By analogy, nanomaterials may also be associated with the same outcomes. Here, the effects of exposure to PM from ambient air, diesel exhaust and certain nanomaterials on atherosclerosis and vasomotor function in animals have been assessed. The majority of studies have used pulmonary exposure by inhalation or instillation, although there are some studies on non-pulmonary routes such as the gastrointestinal tract. Airway exposure to air pollution particles and nanomaterials is associated with similar effects on atherosclerosis progression, augmented vasoconstriction and blunted vasorelaxation responses in arteries, whereas exposure to diesel exhaust is associated with lower responses. At present, there is no convincing evidence of dose-dependent effects across studies. Oxidative stress and inflammation have been observed in the arterial wall of PM-exposed animals with vasomotor dysfunction or plaque progression. From the data, it is evident that pulmonary and systemic inflammation does not seem to be necessary for these vascular effects to occur. Furthermore, there is inconsistent evidence with regard to altered plasma lipid profile and systemic inflammation as a key step in vasomotor dysfunction and progression of atherosclerosis in PM-exposed animals. In summary, the results show that certain nanomaterials, including TiO2, carbon black and carbon nanotubes, have similar hazards to the vascular system as combustion-derived PM.

Synergistic Effects of Zinc Oxide Nanoparticles and Fatty Acids on Toxicity to Caco-2 Cells

Fatty acids exposure may increase sensitivity of intestinal epithelial cells to cytotoxic effects of zinc oxide (ZnO) nanoparticles (NPs). This study evaluated the synergistic effects of ZnO NPs and palmitic acid (PA) or free fatty acids (FFAs) mixture (oleic/PA 2:1) on toxicity to human colon epithelial (Caco-2) cells. The ZnO NPs exposure concentration dependently induced cytotoxicity to Caco-2 cells showing as reduced proliferation and activity measured by 3 different assays. PA exposure induced cytotoxicity, and coexposure to ZnO NPs and PA showed the largest cytotoxic effects. The presence of FFAs mixture did not affect the ZnO NPs-induced cytotoxicity. Filtration of freshly prepared suspension of NPs through a 0.45-µm pore size membrane significantly reduced the cytotoxicity, indicating a role of concentration or size of particles in cytotoxic effects. The ZnO NPs and PA coexposure induced production of mitochondrial reactive oxygen species (mROS) but not intracellular ROS production, whereas FFAs mixture exposure did not induce mROS and inhibited intracellular ROS. Both ZnO NPs and fatty acids (PA and FFAs mixture) promoted lysosomal destabilization, which was not correlated with cytotoxicity. These results indicated that PA can enhance ZnO NPs-induced cytotoxicity probably by the augmentation of mROS production, whereas FFAs mixture did not affect ROS production. Synergistic effects between ZnO NPs and fatty acids may be important when considering NPs toxicity via oral exposure.