Immunotoxicology of titanium dioxide and hydroxylated fullerenes engineered nanoparticles in fish models

Nanoparticles have the potential to cause adverse effects on the fish health, but the understanding of the underlying mechanisms is limited. Major task of this dissertation was to connect gaps in current knowledge with a comprehensive sequence of molecular, cellular and organismal responses toward environmentally relevant concentrations of engineered nanoparticles (titanium dioxide - TiO2 and hydroxylated fullerenes), outlining the interaction with the innate immune system of fish. The research was divided into following steps: 1) create cDNA libraries for the species of fathead minnow (Pimephales promelas); 2) evaluate whether, and how can nanoparticles modulate neutrophil function in P. promelas; 3) determine the changes in expression of standard biomarker genes as a result of nanoparticle treatment; 4) expose the P. promelas to nanoparticles and appraise their survival rate in a bacterial challenge study; 5) assess the impact of nanoparticles on neuro-immunological interface during the early embryogenesis of zebrafish (Danio rerio). It was hypothesized that engineered nanoparticles can cause measurable changes in fish transcriptome, immune response, and disease resistance. The results of this dissertation are: 1) application of environmentally relevant concentration of nanoparticles changed function of fish neutrophils; 2) fish exposed to nano- TiO2 had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2, while expression of interleukin 11 and myeloperoxidase was significantly increased and expression of elastase 2 was significantly decreased in fish exposed to hydroxylated fullerenes; 3) exposure to environmental estimated concentration of nano-TiO2 significantly increased fish mortality during Aeromonas hydrophila challenge. Analysis of nano-TiO2 distribution in fish organism outlined that the nano-TiO2 is concentrating in the fish kidney and spleen; 4) during the early embryogenesis of D. rerio exposure to nanoparticles caused shifts in gene regulation response patterns. Significant effects on gene regulation were observed on genes involved in circadian rhythm, kinase activity, vesicular transport and immune response

Assessment of heavy metal load in chub liver (Cyprinidae – Leuciscus cephalus) from the Nišava River (Serbia)

The Nišava River plays an important role as the source for both drinking water and agricultural irrigation dueto its hydrological and geomorphological characteristics as the largest river in the region of southeast Serbia.In this study we used the liver of the chub (Leiciscus cephalus) as a tool for biomonitoring heavy metalaccumulation along the river. Chub specimens were sampled from two localities (one at the border withBulgaria and a second in the city of Niš). Concentrations were estimated for six heavy metals (iron,cadmium, copper, zinc, lead and manganese) in chub liver. Low bioconcentration level was observed formost of the metals and the concentrations corresponded to the nominal concentration in livers of fishinhabiting metal unpolluted streams and rivers. However, cadmium concentration in the chub liver exceeded0.5 mg kg-1, a several hundred folds increase from nominal concentration indicating a potential toxicexposure of the fish and of the stream ecosystem to this heavy metal. Hepatosomatic indices were calculatedand tested for the impact of metal concentrations on liver size. A decrease of the hepatic index was observedin fish with higher cadmium concentration, suggesting a possible impact on the health of the chub populationin the Nišava River

Titanium dioxide nanoparticles enhance mortality of fish exposed to bacterial pathogens

Nano-TiO2 is immunotoxic to fish and reduces the bactericidal function of fish neutrophils. Here, fathead minnows (Pimephales promelas) were exposed to low and high environmentally relevant concentration of nano-TiO2 (2 ng g−1 and 10 μg g−1 body weight, respectively), and were challenged with common fish bacterial pathogens, Aeromonas hydrophila or Edwardsiella ictaluri. Pre-exposure to nano-TiO2 significantly increased fish mortality during bacterial challenge. Nano-TiO2 concentrated in the kidney and spleen. Phagocytosis assay demonstrated that nano-TiO2 has the ability to diminish neutrophil phagocytosis of A. hydrophila. Fish injected with TiO2 nanoparticles displayed significant histopathology when compared to control fish. The interplay between nanoparticle exposure, immune system, histopathology, and infectious disease pathogenesis in any animal model has not been described before. By modulating fish immune responses and interfering with resistance to bacterial pathogens, manufactured nano-TiO2 has the potential to affect fish survival in a disease outbreak

Histopathology of fathead minnow (Pimephales promelas) exposed to hydroxylated fullerenes

Hydroxylated fullerenes are reported to be very strong antioxidants, acting to quench reactive oxygen species, thus having strong potential for important and widespread applications in innovative therapies for a variety of disease processes. However, their potential for toxicological side effects is still largely controversial and unknown. Effects of hydroxylated fullerenes C60(OH)24 on the fathead minnow (Pimephales promelas) were investigated microscopically after a 72-hour (acute) exposure by intraperitoneal injection of 20 ppm of hydroxylated fullerenes per gram of body mass. Cumulative, semi-quantitative histopathologic evaluation of brain, liver, anterior kidney, posterior kidney, skin, coelom, gills and the vestibuloauditory system revealed significant differences between control and hydroxylated fullerene-treated fish. Fullerene-treated fish had much higher cumulative histopathology scores. Histopathologic changes included loss of cellularity in the interstitium of the kidney, a primary site of haematopoiesis in fish, and loss of intracytoplasmic glycogen in liver. In the coelom, variable numbers of leukocytes, including many macrophages and fewer heterophils and rodlet cells, were admixed with the nanomaterial. These findings raise concern about in vivo administration of hydroxylated fullerenes in experimental drugs and procedures in human medicine, and should be investigated in more detail.This article is published as Jovanović, Boris, Elizabeth M. Whitley, and Dušan Palić. "Histopathology of fathead minnow (Pimephales promelas) exposed to hydroxylated fullerenes." Nanotoxicology 8, no. 7 (2014): 755-763. doi: 10.3109/17435390.2013.828794.</p

The effects of a human food additive, titanium dioxide nanoparticles E171, on Drosophila melanogaster - a 20 generation dietary exposure experiment

In this study, fruit flies (Drosophila melanogaster) were exposed to an estimated daily human E171 consumption concentration for 20 generations. Exposure to E171 resulted in: a change in normal developmental and reproductive dynamics, reduced fecundity after repetitive breeding, increased genotoxicity, the appearance of aberrant phenotypes and morphologic changes to the adult fat body. Marks of adaptive evolution and directional selection were also exhibited. The larval stages were at a higher risk of sustaining damage from E171 as they had a slower elimination rate of TiO2 compared to the adults. This is particularly worrisome, since among the human population, children tend to consume higher daily concentrations of E171 than do adults. The genotoxic effect of E171 was statistically higher in each subsequent generation compared to the previous one. Aberrant phenotypes were likely caused by developmental defects induced by E171, and were not mutations, since the phenotypic features were not transferred to any progeny even after 5 generations of consecutive crossbreeding. Therefore, exposure to E171 during the early developmental period carries a higher risk of toxicity. The fact that the daily human consumption concentration of E171 interferes with and influences fruit fly physiological, ontogenetic, genotoxic, and adaptive processes certainly raises safety concerns

Phytochemistry, Toxicology and Therapeutic Value of Petasites hybridus Subsp. Ochroleucus (Common Butterbur) from the Balkans

Petasites hybridus (Common butterbur) is extensively used in traditional medicine, and is currently gaining interest and popularity as a food supplement and for its medicinal properties. It contains a large number of active compounds of potential therapeutic activity, but also toxic pyrrolizidine alkaloids. Science-based information is needed to support the developing modern use of this plant, and to direct continued safe practice in traditional medicine. The present study focused on the essential oils from leaves and rhizomes of the understudied P. hybridus ssp. ochroleucus from the Balkans, and evaluated its phytochemistry and potential therapeutic activities (antimicrobial, antioxidant, anti-cholinesterase and anti-inflammatory), as well its toxicology potential (acute toxicity in insects and mice). We studied the essential oils, which are not commonly used in traditional practices, but have a potential for safe use since the toxic pyrrolizidine alkaloids, which are non-volatiles, are usually not present in the distilled essential oils. Pyrrolizidine alkaloids were indeed not detected in the essential oils; ingestion of the essential oils did not induce toxicity signs in mice, and topical application did not elicit skin irritation in humans. The essential oils had no antimicrobial properties against 20 pathogenic bacterial strains, but demonstrated good local anti-inflammatory activity in a Carrageenan-induced paw edema test. An insect toxicity test demonstrated that the leaf essential oil is an efficient insect repellent, and the demonstrated anti-cholinesterase activity suggests a potential for the treatment of neurological conditions. Isopetasin, a sesquiterpene found in plants of the genus Petasites, known to have anti-inflammatory effects, was present only in the rhizomes essential oil (3.9%), and sesquiterpene lactones concentrations were high, likely contributing to the antioxidant activity

A multiparametric approach to cerium oxide nanoparticle toxicity assessment in non‐biting midges

Cerium oxide nanoparticles (nano-CeO2) are included in the OECD priority list of engineered nanomaterials for the assessment of their environmental impact. The present study was carried out in order to assess the nano-CeO2 toxicity to freshwater midge Chironomus riparius larvae at concentrations of 2.5, 25, 250 and 2500 mg of nano-CeO2 per kg of sediment. Experiments were designed to assess the prolonged exposure of midges to nano-CeO2 while adhering to OECD guideline number 218. The following parameters were investigated: nano-CeO2 uptake by the larvae, oxidative stress parameters, the in vivo genotoxic effect and life trait parameters. ICP-MS analysis showed a significant positive correlation between the concentration of nano-CeO2 in the sediment and its uptake by the larvae. No significant mortality was observed in C. riparius and oxidative stress was not detected. The only significantly induced sublethal effect was genotoxicity, which began to manifest at a LOEC concentration of 25 mg kg-1 of sediment and progressively increased at higher concentrations. Therefore, this study indicates that exposure to nano-CeO2 contaminated freshwater sediments does not pose a risk to chironomids at environmentally realistic concentrations. However, significant accumulation of nano-CeO2 by chironomid larvae may pose a risk through trophic transfer to organisms further up in the food chain

Nasal morphological characteristics of the serbian population

The aim of this study was to determine the nasal parameters in the population of central Serbia and to compare them with those determined in earlier studies in different populations. The research was conducted on 496 randomly selected persons (262 males and 234 females), aged 18-65 years. The measured parameters were nasal height and nasal breadth and the standard spreading caliper with scale was used for measurements. There were significant differences in the nasal parameters between male and female subjects. The nasal breadth was 34.72 mm in females, and in the male population it was 36.7 mm. The mean values of nasal height were 52.6 mm and 54.32 mm in females and males, respectively. The nasal index in females and males was 66.01 and 67.56, respectively, and the mean value of the nasal index of all respondents was 66.78. After conducting the research it was concluded that the dominant nasal type in the population of the central part of Serbia is leptorrhine. The present study showed the existence of sexual dimorphism in nasal morphology. The data obtained in our study may be useful in anthropological and forensic research, as well as in cosmetic planning and reconstructive surgery

Sexual dimorphism of medium-sized neurons with spines in human nucleus accumbens

The nucleus accumbens is a limbic nucleus, representing part of the striatum body, and together with the caudate nucleus and putamen, it lies on the septum. The aim of this study was to examine morphological sexual dimorphism in spine density and also to undertake an immunohistochemical study of expression for estrogen and progesterone receptors in the medium-sized neurons in the nucleus accumbens. The research was conducted on twenty human brains of persons of both sexes, between the age of 20-75 years. The Golgi method was applied to determine the types and subtypes of neurons, morphologies of soma, dendrites and axons, as well as the relations between the cells and glial elements. The following were quantitatively examined: the maximum diameter of the neurons, the minimal diameter of the neurons, and the total length of the dendrites. The expression of receptors for estrogen and progesterone, their distribution and intensity were defined immunohistochemically. The parameters of the bodies of neurons in the shell and core of the nucleus accumbens were studied in both men and women. No statistically significant differences were found. Examination of the spine density showed statistical significance in terms of a higher density of spines in women. Immunohistochemically, in the female brain estrogen expression is diffusely spread in a large number of neurons; it is extra nuclear, of granular appearance and high intensity. In the male brain, expression of estrogen is visible and distributed over about one half of different types of neurons; it is extra nuclear, of granular appearance, mostly of middle and low staining intensity. Expression of progesterone in the female brain was very discreet and on a very small number of neurons; it was extra nuclear and with a weak staining intensity. Expression of progesterone in the male brain was distributed on a small number of neurons. It had a granular appearance, it was extra nuclear, with a very low staining intensity. Our results show differences in the morphology as well as expression of receptors for estrogen and progesterone on medium-sized neurons with spines in the nucleus accumbens of men and women

Anthropometric study of the facial index in the population of Central Serbia

The aim of this study was to determine the craniofacial parameters in the population of the central part of Serbia. The research was conducted on 700 persons (360 males and 340 females), aged 18-65 years, selected randomly. The measured parameters were morphological facial height and breadth. The standard spreading caliper with scale was used for the measurement of facial parameters. There were significant differences in the facial parameters of male compared to female subjects in all observed parameters. The mean value of the morphological facial height in the study population was 116.8 mm ± 7.28, maximum facial breadth 124.12 mm ± 8.44, while the mean value of the total facial index was 93.68 ± 6.86. The total facial index was calculated according to the formula and the obtained results were analyzed statistically using the t-test. The dominant phenotype in the studied population was leptoprosopic. The data obtained in our study may be useful in anthropological research, forensics, genetic research, as well as in medical clinical practice