Impact of acute and subchronic inhalation exposure to PbO nanoparticles on mice.

Lead nanoparticles (NPs) are released into air from metal processing, road transport or combustion processes. Inhalation exposure is therefore very likely to occur. However, even though the effects of bulk lead are well known, there is limited knowledge regarding impact of Pb NPs inhalation. This study focused on acute and subchronic exposures to lead oxide nanoparticles (PbO NPs). Mice were exposed to PbO NPs in whole body inhalation chambers for 4-72 h in acute experiment (4.05 × 106 PbO NPs/cm3), and for 1-11 weeks in subchronic experiment (3.83 × 105 particles/cm3 in lower and 1.93 × 106 particles/cm3 in higher exposure group). Presence of NPs was confirmed in all studied organs, including brain, which is very important considering lead neurotoxicity. Lead concentration gradually increased in all tissues depending on the exposure concentration and duration. The most burdened organs were lung and kidney, however liver and brain also showed significant increase of lead concentration during exposure. Histological analysis documented numerous morphological alterations and tissue damage, mainly in lung, but also in liver. Mild pathological changes were observed also in kidney and brain. Levels of glutathione (reduced and oxidized) were modulated mainly in lung in both, acute and subchronic exposures. Increase of lipid peroxidation was observed in kidney after acute exposure. This study characterized impacts of short to longer-term inhalation exposure, proved transport of PbO NPs to secondary organs, documented time and concentration dependent gradual increase of Pb concentration and histopathological damage in tissues.

Impact of acute and chronic inhalation exposure to CdO nanoparticles on mice.

Cadmium nanoparticles can represent a risk in both industrial and environmental settings, but there is little knowledge on the impacts of their inhalation, especially concerning longer-term exposures. In this study, mice were exposed to cadmium oxide (CdO) nanoparticles in whole body inhalation chambers for 4 to 72 h in acute and 1 to 13 weeks (24 h/day, 7 days/week) in chronic exposure to investigate the dynamics of nanoparticle uptake and effects. In the acute experiment, mice were exposed to 2.95 × 106 particles/cm3 (31.7 µg CdO/m3). The same concentration and a lower one (1.18 × 106 particles/cm3, 12.7 µg CdO/m3) were used for the chronic exposure. Transmission electron microscopy documented distribution of nanoparticles into all studied organs. Major portion of nanoparticles was retained in the lung, but longer exposure led to a greater relative redistribution into secondary organs, namely the kidney, and also the liver and spleen. Accumulation of Cd in the lung and liver occurred already after 24 h and in the brain, kidney, and spleen after 72 h of exposure, and a further increase of Cd levels was observed throughout the chronic exposure. There were significant differences in both Cd accumulation and effects between the two exposure doses. Lung weight in the higher exposure group increased up to 2-fold compared to the control. Histological analyses showed dose-dependent alterations in lung and liver morphology and damage to their tissue. Modulation of oxidative stress parameters including glutathione levels and increased lipid peroxidation occurred mainly after the greater chronic exposure. The results emphasize risk of longer-term inhalation of cadmium nanoparticles, since adverse effects occurring after shorter exposures gradually progressed with a longer exposure duration.

Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles.

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.

Expression, function and regulation of Evi-1 during embryonic avian development.

Ecotropical viral integration site 1 (Evi-1) is a transcription factor essential for vascularisation and cell proliferation during embryonic development. The chimeric transcription factor AML1-EVI-1 is activated in leukaemia where it plays a role as a differentiation block and stimulator of proliferation. Here, we cloned chicken Evi-1 and analysed its expression during embryonic development. There was early expression in the pharyngeal arches, in the brain and intermediate mesoderm of chicken embryos at stage 15. Later at stage 20, Evi-1 mesenchymal expression was concentrated in the second pharyngeal arch, and weaker expression was found in the mandibular and maxillary prominences. Facial expression decreased in intensity during development. Evi-1 expression in the limb was also limited to the mesenchyme with the most prominent expression in the anterior margin. Evi-1 was not detectable in the posterior limb bud. At later stages, Evi-1 was expressed in the peripheral mesenchyme of the limb but not in the developing precartilage blastema. At stage 29, the expression became restricted to the perichondrium and interdigital areas; however, the cartilage condensations themselves were negative. To study the function of Evi-1 in chondrogenesis, we knocked down expression in limb micromass cultures using siRNA. Chondrogenesis was significantly reduced in both anterior and posterior cultures. Since Evi-1 was expressed adjacent to the apical ectodermal ridge and this area is a source of FGFs, we tested whether endogenous FGF receptor signalling was necessary to maintain its expression. Inhibitors of FGFRs (PD161570 and SU5402) were applied to wing mesenchyme, and downregulation of Evi-1 expression was observed after treatment with both inhibitors. Therefore, Evi-1 may be a transcription factor mediating the effects of FGF and may also be defining the size of cartilage elements in the limb.

Investigation of an autologous blood treatment strategy for temporomandibular joint hypermobility in a pig model.

Many different surgical and non-surgical techniques are used for the treatment of temporomandibular joint (TMJ) hypermobility. One of these methods is autologous blood injection into the TMJ. The fate of the autologous blood used for treatment of recurring condylar dislocation is still not completely understood. The authors used 12 pigs (Sus scrota f. domestica) as a model species for autologous blood delivery into the TMJ. Blood injection was followed by histopathological analysis at different times after treatment (1h, 1, 2 and 4 weeks). Samples were examined by magnetic resonance imaging, macroscopic and histological methods. The deposition of the remaining blood was observed in the form of clots in the distal parts of the upper joint cavity 1h and 1 week after treatment. 2 weeks after treatment, small blood clots were still apparent in the distal part of the upper joint cavity. 4 weeks after surgery, no remnants of blood, changes or adhesions were apparent inside the TMJ. No morphological or histological changes were observed in the TMJ after the injection of autologous blood suggesting another mechanism is involved in the hypermobility treatment.

Formation of the tooth-bone interface.

Not only are teeth essential for mastication, but also missing teeth are considered a social handicap due to speech and aesthetic problems, with a resulting high impact on emotional well-being. Several treatment procedures are currently available for tooth replacement with mostly inert prosthetic materials and implants. Natural tooth substitution based on copying the developmental process of tooth formation is particularly challenging and creates a rapidly developing area of molecular dentistry. In any approach, functional interactions among the tooth, the surrounding bone, and the periodontium must be established. Therefore, recent research in craniofacial genetics searches for mechanisms responsible for correct cell and tissue interactions, not only within a specific structure, but also in the context of supporting structures. A tooth crown that is not functionally anchored to roots and bone is useless. This review aims to summarize the developmental and tissue homeostatic aspects of the tooth-bone interface, from the initial patterning toward tooth eruption and lifelong interactions between the tooth and its surrounding alveolar bone.

Bendiocarbamate toxicity in the chick embryo.

Carbamate pesticides generally possess low toxicity for warm-blooded vertebrates, but developmental data are scarce. We have therefore evaluated embryotoxicity of choline esterase inhibitor bendiocarbamate in the chick embryo. The pesticide was dissolved in 5% acetone in distilled water and a volume of 200 microl was administered over the embryo through membrana papyracea on embryonic days 2, 3, 4, 5, and 10. Sampling was performed on embryonic day 10, while the embryos treated on embryonic day 10 were sampled on embryonic day 17. The toxicity of bendiocarbamate was fairly low, and LD50 decreased with advancing development from 1 mg/ embryo on embryonic day 2 to 29 mg on embryonic day 5. Malformations in surviving embryos were observed rarely (< 3 %) and occurred in both control and experimental groups. There was a mild but statistically significant dose-dependent reduction in body weight, most pronounced in the treatment on embryonic days 5 and 10, but the maximum difference from controls was below 15 %. A small but not significant increase in the number of positive cells was observed in the eye, limb buds, and the central nervous system of embryos treated on embryonic days 3 and 4 and examined after supravital whole-mount staining with Lysotracker Red for apoptosis. In agreement with previously published studies in other vertebrate animals, we conclude that bendiocarbamate does not possess significant toxicity in the avian embryo.

Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice.

During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

CD 95 mediated apoptosis in embryogenesis: implication in tooth development.

INTRODUCTION: Understanding of apoptotic mechanisms involved in tissue shaping is of particular interest because of possible targeted modulation of the development of organ structures such as teeth. Research of CD 95 mediated apoptosis has been focused particularly on cell death in the immune system and related disorders. However, CD 95 mediated apoptosis is also involved in embryogenesis of many organs as the kidney, the lung, the intestine and tissue networks such as the nervous system. DESIGN: Narrative review. RESULTS: This review briefly summarizes the current knowledge of CD 95 mediated apoptosis in embryogenesis with possible implication in tooth development. CD 95 receptor and CD 95 ligand are found at early stages of tooth development. The data suggest some positive correlations with dental apoptosis distribution, particularly in the primary enamel knot where apoptosis occurs during elimination of this structure. CD 95 deficient (lpr) adult mouse tooth phenotype, however, did not show any alterations in final tooth pattern and morphology. CONCLUSION: To date studies of apoptotic machinery during tooth development show spatial localization of many of the components together with precise and localized timing of cell death. There is still much to be learned about the regulation and importance of apoptosis in tooth development. Nevertheless, the involvement of apoptotic regulatory mechanisms interplaying with other molecules participates to the cellular cross-talk in developing tissues, which opens possible targeted modulations as suggested, e.g. for future molecular dentistry.

Caspase 3 activation in the primary enamel knot of developing molar tooth.

Mammalian teeth develop during embryogenesis as epithelio-mesenchymal organs. The primary enamel knot is considered as a signaling center in tooth morphogenesis. After tooth bell formation, this epithelial structure undergoes apoptosis. Activation of caspase 3 represents a crucial step in the intracellular death machinery. Procaspase 3 and caspase 3 molecules were localized in the primary enamel knot of the field vole using immunohistochemistry. Different fixation procedures in cryopreserved and paraffin-embedded tissues and detection systems based on peroxidase and alkaline phosphatase mediated color reactions were applied. Apoptosis was detected using morphological criteria and the TUNEL assay. Procaspase 3 was found in both the epithelial and mesenchymal part of the tooth germ. Active caspase 3 was localized particularly in the primary enamel knot, its distribution correlated with dental apoptosis and showed a similar pattern in the field vole as in the mouse.

Apoptosis-related factors (Fas receptor, Fas ligand, FADD) in early tooth development of the field vole (Microtus agrestis).

Fas (CD95/APO-1) belongs to the TNF receptor (TNFR) family. Fas ligand binding followed by Fas-receptor oligomerisation leads to formation of a death-inducing signal complex starting with recruitment of the Fas-adapter protein (FADD). Components of this initiation complex (Fas, Fas-L, FADD) were correlated with apoptotic cells, detected by specific DNA fragmentation and morphological criteria. Apoptotic cells can be detected throughout the embryonic development of molar teeth. Restricted temporospatial distribution suggests several important roles for apoptosis in tooth morphogenesis. However, the mechanisms employed in dental apoptosis remain unclear. Frontal sections of the field vole at stage 13.5-15.5 of embryonic development were exploited to investigate and correlate location of Fas, Fas-ligand, FADD molecules and apoptosis in developing first molars by immunohistochemistry. During these stages the primary enamel knot appears and is gradually terminated by apoptosis. Initially, apoptotic cells were demonstrated in the most superficial layer of the dental lamina. The number of TUNEL-positive cells expanded from late bud to cap stages. Restricted areas of apoptotic cells were found in the stalk and primary enamel knot. Fas, Fas-L and FADD were co-localised, particularly in the primary enamel knot, and the stalk, correlating with the occurrence of apoptosis in these areas. Fas-L, however, was also found in proliferating parts of the developing tooth germ, such as in the cervical loops. Interestingly, FADD molecules were also observed in areas, where Fas protein was not detected. According to the immunohistochemical data, Fas-mediated signalling may have a triggering or enhancing role in dental apoptosis. This remains to be functionally confirmed.

Apoptosis distribution in the first molar tooth germ of the field vole (Microtus agrestis).

Apoptosis represents an important process in organ and tissue morphogenesis and remodeling during embryonic development. A role for apoptosis in shape formation of developing teeth has been suggested. The field vole is a useful model for comparative studies in odontogenesis, particularly because of its contrasting molar morphogenesis when compared to the mouse. However, little is known concerning apoptosis in tooth development of this species. Morphological (cellular and nuclear alterations) and biochemical (specific DNA breaks--TUNEL staining) characteristics of apoptotic cells were used to evaluate the temporal and spatial occurrence of apoptosis in epithelial and mesenchymal tissues of the developing first molar tooth germs of the field vole. Apoptotic cells were found in non-proliferating areas (identified previously) throughout bud to bell stages, particularly in the epithelium, however, scattered also in the mesenchyme. A high concentration of TUNEL positive cells was evident in primary enamel knots at late bud stage with increasing density of apoptotic cells until ED 16 when the primary enamel knot in the field vole disappears and mesenchyme becomes protruded in the middle axes of the bell forming two shallow areas with zig-zag located secondary enamel knots. Distribution of TUNEL positive cells corresponded with localisation of secondary enamel knots as shown using histological and 3D analysis. Apoptosis was shown to be involved in the first molar development of the field vole, however, exact mechanisms and roles of this process in tooth morphogenesis require further investigation.

A striated muscle on the hard palate of rodents and rabbits.

Summary A striated muscle of the hard palate has been previously described in some rodents and rabbits. It is not termed in the official veterinary anatomical nomenclature. The aim of this work was to verify the existence of this muscle. Heads of the golden hamster (Mesocricetus auratus), the guinea pig (Cavia aperea f. porcellus), the laboratory rat (Rattus norvegicus var. alba), the field vole (Microtus agrestis) and the domestic rabbit (Oryctolagus cuniculus f. domesticus) have been dissected. Moreover, histological sections have been prepared from heads of the field vole. In all species under study, we could detect a striated muscle of the hard palate composed of an anterior and a posterior muscle. The anterior muscle originated on the os incisivum and diverged in anterior, lateral and posterior directions. The posterior muscle originated on the processus palatinus maxillae and verged into the m. buccinator. Inter-species differences could be detected in shape and position of the muscle. The palatal muscle was innervated by the ramus buccalis of the facial nerve. Whether this muscle should be classified as an individual facial muscle or as a part of the m. buccinator remains to be discussed.

The lateral enamel lamina--component of tooth primordia in selected mammalian species.

The lateral enamel lamina (LEL) is a part of the enamel organ, which is probably not involved in tooth formation. It represents, besides the "stalk" of the tooth primordium, a second interconnection between enamel organ and oral epithelium or vestibular lamina. We detected the LEL in the sheep (Ovis aries), the dolphin (Stenella attenuata), and the vole (Microtus agrestis) by light microscopy and computer-aided three-dimensional reconstruction. The LEL could be found in cap to bell stage tooth primordia, most clearly in slowly developing tooth germs. LEL-like structures have been furthermore described or depicted in tooth germs of the mouse, the elk (Alces alces), the dugong (Dugong dugong), the elephant (Loxodonta africana), and the human. Probably it is a part of all mammalian tooth primordia that undergoes regression during morphogenesis of the enamel organ. As a reducing structure, it should be considered in studies of tooth development.

Proliferating cell nuclear antigen (PCNA) expression in tooth primordia in the field vole (Microtus agrestis, Rodentia).

Cell proliferation in developing tooth germs has been studied particularly using bromodeoxyuridine (BrdU) incorporation into growing tooth primordia and by counting and three-dimensional (3D) reconstruction of mitoses in serial sections of developing teeth. PCNA has been proposed as an alternative marker of proliferation activity. The aim of our study was to detect immunohistochemically locations of PCNA-positive cells in developing tooth germs of Microtus agrestis (Rodentia). PCNA expression could be distinguished in oral epithelium and mesenchyme before first signs of dental lamina elevation. During bud, cap, and bell stages, positive immunostaining could be observed at defined sites in enamel organ, tooth papilla, and dental follicle. Rudimental tooth germs of the upper diastema, enamel knots, and inner enamel epithelium at day of ontogeny 18 and 19 showed negative reaction. PCNA marks cycling and early G0 cells and can be used successfully as a proliferation marker even in collection material.

The effects of two different decalcification procedures on size and structure of embryonic epithelial tissue in objects prepared for light microscopy.

Electrolytic decalcification is a very fast and effective method for removing calcium compounds from bones with minimum damage to tissues. Changes of dimension of tissues in histological sections prepared from specimens decalcified by immersion in a formic acid solution and sections prepared from specimens treated in an electrolytic decalcifier were studied. Heads of mouse foetuses were cut in half, decalcified by one of the above-mentioned methods and embedded in histowax. Dimensional changes of skin, tongue and nasal epithelia in histological sections were evaluated by t-test. Significant shrinking and other unwanted effects of decalcification, such as acidophilia of nuclei, were found in objects decalcified by both methods. No significant differences in the effects of the two methods on tissue dimensions were demonstrated. It is concluded that both decalcification methods are equivalent from the qualitative point of view.

On the development of Cetacean extremities: II. Morphogenesis and histogenesis of the flippers in the spotted dolphin (Stenella attenuata).

Externally, the flippers of Cetacea resemble fish fins, but their internal structure is entirely mammalian. They show, however, some adaptative deviations from the typical pattern of the mammalian extremities, the most striking of which is an increased number of phalanges. The aim of this study is to describe the course of the development of flippers in the spotted dolphin (Stenella attenuata) and compare its features with other similar species from an evolutionary perspective. Early stages of flipper development were studied histologically. Differentiation of cartilaginous anlagens of the skeleton progresses proximodistally, condensation in digital rays being evident sooner than chondrogenesis in the carpal region. In one specimen, the temporary presence of cartilaginous rudiments of two carpal elements, which are not found in adults, was observed. At all examined stages, phalangeal number progressively increases up to (radial to ulnar) 3, 7, 7, 5, 3 in the most advanced stage. The reason for this condition is the specialised function of these limb-like structures. It is a classical example of convergence, in which mammalian extremities change their form to emulate the fin function. A similar condition is found in another group of originally terrestrial animals secondarily fully adapted to the aquatic mode of life-Ichyosauria (Reptilia).

On the development of Cetacean extremities: I. Hind limb rudimentation in the Spotted dolphin (Stenella attenuata).

The Cetacea are group of animals which have completely lost their hind limbs during the course of evolution as a result of their entirely aquatic mode of life. It is known, however, that during their embryonal period, the hind limb buds are temporarily present. The control mechanisms of this regression are not yet understood, and vestigial limbs can sometimes be found in adults. The aim of the present study is to describe the course of hind limb rudimentation during prenatal development of Stenella attenuata (Spotted dolphin) at tissue and cell levels and compare the results with other natural or experimentally induced amelias. Hind limb buds of dolphin embryos, CRL 10-30 mm, were examined histologically. Before total disappearance, they show histodifferentiation comparable with other mammals. Initially, they form the apical ectodermal ridge, which soon regresses. The mesenchyme undergoes the process of condensation to form anlagens of prospective skeletal elements. These condensations are surrounded by vascular plexuses. During the course of rudimentation, some mesenchymal cells die, while the others are incorporated into the body wall. Nerve ingrowth into rudimentary limb buds was also detected. The temporary presence of hind limb rudiments in cetacean embryos can be regarded as a good example of recapitulation of phylogenesis in ontogenesis.

Karyotype of the giant mole-rat, Cryptomys mechowi (Rodentia, Bathyergidae).

The karyotype of the giant mole-rat, Cryptomys mechowi (Rodentia, Bathyergidae), from Zambia was investigated in one male and one female by means of G-, C-, and AgNOR-banding techniques. The diploid chromosomal set consisted of 40 biarmed chromosomes (2n = 40, NF = 80). A pair of autosomes in the male and the X chromosomes in the female were heteromorphic. The sex chromosomes were unusually large.

Sex chromosome abnormalities in natural populations of the common vole (Microtus arvalis).

Four specimens with an aberrant sex chromosome constitution were found in natural populations of the common vole (Microtus arvalis). Two females had an X0 sex chromosome constitution and single males were 2n = 47, XXY and 2n = 47, XYY, respectively. No apparent phenotypical anomalies were recorded in the sex chromosome aneuploids, but their fertility may have been impaired. The incidence of sex chromosome aneuploidy seems to be unusually high in natural populations of the common vole (1.5% of animals examined). Possible explanations for this are discussed.