Multiwalled Carbon Nanotubes Induce Fibrosis and Telomere Length Alterations.

Telomere shortening can result in cellular senescence and in increased level of genome instability, which are key events in numerous of cancer types. Despite this, few studies have focused on the effect of nanomaterial exposure on telomere length as a possible mechanism involved in nanomaterial-induced carcinogenesis. In this study, effects of exposure to multiwalled carbon nanotubes (MWCNT) on telomere length were investigated in mice exposed by intrapleural injection, as well as in human lung epithelial and mesothelial cell lines. In addition, cell cycle, apoptosis, and regulation of genes involved in DNA damage repair were assessed. Exposure to MWCNT led to severe fibrosis, infiltration of inflammatory cells in pleura, and mesothelial cell hyperplasia. These histological alterations were accompanied by deregulation of genes involved in fibrosis and immune cell recruitment, as well as a significant shortening of telomeres in the pleura and the lung. Assessment of key carcinogenic mechanisms in vitro confirmed that long-term exposure to the long MWCNT led to a prominent telomere shortening in epithelial cells, which coincided with G1-phase arrest and enhanced apoptosis. Altogether, our data show that telomere shortening resulting in cell cycle arrest and apoptosis may be an important mechanism in long MWCNT-induced inflammation and fibrosis.

Fetal adrenal development: comparing effects of combined exposures to PCB 118 and PCB 153 in a sheep model.

This study investigated the effects of exposure to the ubiquitous contaminants polychlorinated biphenyls (PCBs) on the fetal adrenal cortex and on plasma cortisol using the domestic sheep (Ovis aries) as a model. Pregnant ewes were intendedly subjected to oral treatment with PCB 153 (98 µg/kg bw/day), PCB 118 (49 µg/kg bw/day) or the vehicle corn oil from mating until euthanasia on gestation day 134 (±0.25 SE). However, because of accidental cross-contamination occurring twice causing a mixed exposure scenario in all three groups, the focus of this paper is to compare three distinct groups of fetuses with different adipose tissue PCB levels (PCB 153high, PCB 118high and low, combined groups) rather than comparing animals exposed to single PCB congeners to those of a control group. When comparing endocrine and anatomical parameters from fetuses in the PCB 153high (n = 13) or PCB 118high (n = 14) groups with the low, combined group (n = 14), there was a significant decrease in fetal body weight (P < 0.05), plasma cortisol concentration (P < 0.001) and adrenal cortex thickness (P < 0.001). Furthermore, adrenal weight was decreased and plasma ACTH was increased only in the PCB 118high group. Expression of several genes encoding enzymes and receptors related to steroid hormone synthesis was also affected and mostly down-regulated in fetuses with high PCB tissue levels. In conclusion, we suggest that mono-and di-ortho PCBs were able to interfere with growth, adrenal development and cortisol production in the fetal sheep model. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.

In utero and lactational exposure to PCB 118 and PCB 153 alter ovarian follicular dynamics and GnRH-induced luteinizing hormone secretion in female lambs.

The effects of in utero and lactational exposure to two structurally different polychlorinated biphenyl (PCB) congeners on follicular dynamics and the pituitary-gonadal axis in female lambs were investigated. Pregnant ewes received corn oil, PCB 118, or PCB 153, and offspring was maintained until 60 days postpartum. Ovarian follicles were quantified using stereology. Plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured using radioimmunoassay before and after administration of a gonadotropin releasing hormone (GnRH) analog. PCB 118 exposure increased numbers of transitional, secondary, and the sum of secondary, early antral, and antral (Σsecondary-antral) follicles, PCB 153 exposure only increased the number of primary follicles. GnRH-induced LH levels were significantly elevated in the PCB 153 exposure group. We conclude that PCB 153 and PCB 118 alter follicular dynamics in lambs and modulate the responsiveness of the pituitary gland to GnRH.

Exposure to a human relevant mixture of persistent organic pollutants or to perfluorooctane sulfonic acid alone dysregulates the developing cerebellum of chicken embryo.

Prenatal exposure to persistent organic pollutants (POPs) is associated with neurodevelopmental disorders. In the present study, we explored whether a human-relevant POP mixture affects the development of chicken embryo cerebellum. We used a defined mixture of 29 POPs, with chemical composition and concentrations based on blood levels in the Scandinavian population. We also evaluated exposure to a prominent compound in the mixture, perfluorooctane sulfonic acid (PFOS), alone. Embryos (n = 7-9 per exposure group) were exposed by injection directly into the allantois at embryonic day 13 (E13). Cerebella were isolated at E17 and subjected to morphological, RNA-seq and shot-gun proteomics analyses. There was a reduction in thickness of the molecular layer of cerebellar cortex in both exposure scenarios. Exposure to the POP mixture significantly affected expression of 65 of 13,800 transcripts, and 43 of 2,568 proteins, when compared to solvent control. PFOS alone affected expression of 80 of 13,859 transcripts, and 69 of 2,555 proteins. Twenty-five genes and 15 proteins were common for both exposure groups. These findings point to alterations in molecular events linked to retinoid X receptor (RXR) signalling, neuronal cell proliferation and migration, cellular stress responses including unfolded protein response, lipid metabolism, and myelination. Exposure to the POP mixture increased methionine oxidation, whereas PFOS decreased oxidation. Several of the altered genes and proteins are involved in a wide variety of neurological disorders. We conclude that POP exposure can interfere with fundamental aspects of neurodevelopment, altering molecular pathways that are associated with adverse neurocognitive and behavioural outcomes.

Induction of colorectal carcinogenesis in the C57BL/6J and A/J mouse strains with a reduced DSS dose in the AOM/DSS model.

BACKGROUND: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide and thus mouse models of CRC are of significant value to study the pathogenesis. The Azoxymethane/Dextran sulfate sodium (AOM/DSS) model is a widely used, robust initiation-promotion model for chemical induction of colitis-associated CRC in rodents. However, the dosage of chemicals, treatment regimens and outcome measures vary greatly among studies employing this model. Thus, the aim of this study was to examine an AOM/DSS model involving a reduced (1%) dose of DSS for induction of carcinogenesis in A/J and C57BL/6J (B6) mice. RESULTS: We show that colonic preneoplastic lesions can be reliably detected in A/J and B6 mice by use of a AOM/DSS model involving a single injection of 10 mg/kg AOM followed by three 7-day cycles of a low-dose (1%) DSS administration. Supporting existing evidence of A/J mice exhibiting higher susceptibility to AOM than B6 mice, our AOM/DSS-treated A/J mice developed the highest number of large colonic lesions. Clinical symptoms in both strains subjected to the AOM/DSS treatment did not persist in-between treatment cycles, demonstrating that the animals tolerated the treatment well. CONCLUSIONS: Our findings suggest that a reduced dose of DSS in the AOM/DSS model can be considered in future studies of early phase colorectal carcinogenesis in the A/J and B6 mouse strains using preneoplastic lesions as an outcome measure, and that such regimen may reduce the risk of early trial terminations to accommodate human endpoints. Overall, our data emphasize the importance of devoting attention towards choice of protocol, outcome measures and mouse strain in studies of CRC in mice according to the study purpose.

Perinatal exposure to a human relevant mixture of persistent organic pollutants: Effects on mammary gland development, ovarian folliculogenesis and liver in CD-1 mice.

The ability of persistent organic pollutants (POPs) with endocrine disrupting properties to interfere with the developing reproductive system is of increasing concern. POPs are transferred from dams to offspring and the high sensitivity of neonates to endocrine disturbances may be caused by underdeveloped systems of metabolism and excretion. The present study aimed to characterize the effect of in utero and lactational exposure to a human relevant mixture of POPs on the female mammary gland, ovarian folliculogenesis and liver function in CD-1 offspring mice. Dams were exposed to the mixture through the diet at Control, Low or High doses (representing 0x, 5000x and 100 000x human estimated daily intake levels, respectively) from weaning and throughout mating, gestation, and lactation. Perinatally exposed female offspring exhibited altered mammary gland development and a suppressed ovarian follicle maturation. Increased hepatic cytochrome P450 enzymatic activities indirectly indicated activation of nuclear receptors and potential generation of reactive products. Hepatocellular hypertrophy was observed from weaning until 30 weeks of age and could potentially lead to hepatotoxicity. Further studies should investigate the effects of human relevant mixtures of POPs on several hormones combined with female reproductive ability and liver function.

Assessment of cardiac structure and function in a murine model of temporal lobe epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is a significant cause of premature seizure-related death. An association between SUDEP and cardiac remodeling has been suggested. However, whether SUDEP is a direct consequence of acute or recurrent seizures is unsettled. The purpose of this study was to evaluate the impact of status epilepticus (SE) and chronic seizures on myocardial structure and function. We used the intracortical kainate injection model of temporal lobe epilepsy to elicit SE and chronic epilepsy in mice. In total, 24 C57/BL6 mice (13 kainate, 11 sham) were studied 2 and 30 days post-injection. Cardiac structure and function were investigated in-vivo with a 9.4 T MRI, electrocardiography (ECG), echocardiography, and histology [Haematoxylin/Eosin (HE) and Martius Scarlet Blue (MSB)] for staining of collagen proliferation and fibrin accumulation. In conclusion, we did not detect any significant changes in cardiac structure and function neither in mice 2 days nor 30 days post-injection.

Maternal exposure to a human relevant mixture of persistent organic pollutants reduces colorectal carcinogenesis in A/J Min/+ mice.

An increased risk of developing colorectal cancer has been associated with exposure to persistent organic pollutants (POPs) and alteration in the gut bacterial community. However, there is limited understanding about the impact of maternal exposure to POPs on colorectal cancer and gut microbiota. This study characterized the influence of exposure to a human relevant mixture of POPs during gestation and lactation on colorectal cancer, intestinal metabolite composition and microbiota in the A/J Min/+ mouse model. Surprisingly, the maternal POP exposure decreased colonic tumor burden, as shown by light microscopy and histopathological evaluation, indicating a restriction of colorectal carcinogenesis. 1H nuclear magnetic resonance spectroscopy-based metabolomic analysis identified alterations in the metabolism of amino acids, lipids, glycerophospholipids and energy in intestinal tissue. In addition, 16S rRNA sequencing of gut microbiota indicated that maternal exposure modified fecal bacterial composition. In conclusion, the results showed that early-life exposure to a mixture of POPs reduced colorectal cancer initiation and promotion, possibly through modulation of the microbial and biochemical environment. Further studies should focus on the development of colorectal cancer after combined maternal and dietary exposures to environmentally relevant low-dose POP mixtures.

Transcriptional regulation in liver and testis associated with developmental and reproductive effects in male zebrafish exposed to natural mixtures of persistent organic pollutants (POP).

Persistent organic pollutants (POP) occur as mixtures in nature and it is difficult to predict the toxicity of such mixtures based on knowledge about toxicity and mechanisms of action for single compounds. The present knowledge on the combined toxic effects and modes of actions of exposure to mixtures is limited. Thus, the scientifically based hazard and risk assessment of POP requires analytical and toxicological data from studies with environmental mixtures of POP. The application of genome wide transcription profiling in toxicology, in combination with classical endpoints, will improve the current understanding of the mechanisms of toxic processes. Furthermore, gene expression data may be useful in establishing new hypothesis and discovering new biomarkers for known toxicity as well as not yet recognized toxicity endpoints. In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of POP were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBD), polychlorinated biphenyls (PCB), and DDT. The concentration of POP in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa, to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) reduced survival, (2) earlier onset of puberty, (3) increased male/female sex ratio, and (4) differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes included (1) key regulator genes for steroid and thyroid hormone functions (cga, ncoa3), (2) insulin signaling and metabolic homeostasis (pik3r1, pfkfb3, ptb1), and (3) p53 activation (mdm4). The effects observed in the experimental zebrafish model raise the question of whether chemical pollution represents a risk to the reproductive health of wild fish inhabiting the freshwater system.

Phenotypic characterisation of intestinal dendritic cells in sheep.

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.

Long-term levetiracetam treatment affects reproductive endocrine function in female Wistar rats.

PURPOSE: Several antiepileptic drugs (AEDs) induce changes in endocrine function in women with epilepsy. Levetiracetam (LEV) is one of the newer AEDs, and to date no endocrine side-effects have been reported in humans. However, a recent study on ovarian follicular cells from prepubertal pigs showed that LEV affected basal steroid hormone secretion. The aim of the present study was to investigate possible effects of the drug on endocrine function and ovarian morphology in non-epileptic rats. METHODS: Thirty female Wistar rats were fed per-orally with either 50mg/kg LEV (n=15) or 150 mg/kg LEV (n=15) twice daily for 90-95 days. Twenty rats received a control solution. The rats were killed in the dioestrus phase of the oestrous cycle. Serum concentrations of testosterone, 17beta-oestradiol, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH) and LEV were measured, and the ovaries examined histologically. RESULTS: Mean ovarian weight showed a significant, dose-dependent increase after LEV treatment. Mean numbers of ovarian follicular cysts were not changed, but the numbers of corpora lutea and secondary follicles were significantly higher in the treated animals. Serum testosterone was significantly increased in treated animals (0.50 nmol/l versus 0.16 nmol/l in controls, p<0.05), while oestradiol was reduced (67.4 compared to 257.5 pmol/l in controls, p<0.05). The low-dose group had significantly lower serum progesterone concentrations than the control group (56.8 nmol/l versus 34.7 nmol/l, respectively, p<0.05). FSH was reduced in the treated animals (3.3 ng/ml versus 5.5 ng/ml, p<0.05) while LH was unaffected. CONCLUSION: Our findings indicate a possible effect of LEV on the hypothalamic-pituitary-gonadal (HPG) axis and ovarian morphology in non-epileptic rats. The effects differ from those previously described for other AEDs. Caution must be taken before these results can be applied to humans.

Inflammation in the pleural cavity following injection of multi-walled carbon nanotubes is dependent on their characteristics and the presence of IL-1 genes.

Upon inhalation, multi-walled carbon nanotubes (MWCNTs) may reach the subpleura and pleural spaces, and induce pleural inflammation and/or mesothelioma in humans. However, the mechanisms of MWCNT-induced pathology after direct intrapleural injections are still only partly elucidated. In particular, a role of the proinflammatory interleukin-1 (IL-1) cytokines in pleural inflammation has so far not been published. We examined the MWCNT-induced pleural inflammation, gene expression abnormalities, and the modifying role of IL-1α and ß cytokines following intrapleural injection of two types of MWCNTs (CNT-1 and CNT-2) compared with crocidolite asbestos in IL-1 wild-type (WT) and IL-1α/ß KO (IL1-KO) mice. Histopathological examination of the pleura 28 days post-exposure revealed mesothelial cell hyperplasia, leukocyte infiltration, and fibrosis occurring in the CNT-1 (Mitsui-7)-exposed group. The pleura of these mice also showed the greatest changes in mRNA and miRNA expression levels, closely followed by CNT-2. In addition, the CNT-1-exposed group also presented the greatest infiltrations of leukocytes and proliferation of fibrous tissue. WT mice were more prone to development of sustained inflammation and fibrosis than IL1-KO mice. Prominent differences in genetic and epigenetic changes were also observed between the two genotypes. In conclusion, the fibrotic response to MWCNTs in the pleura depends on the particles' physico-chemical properties and on the presence or absence of the IL-1 genes. Furthermore, we found that CNT-1 was the most potent inducer of inflammatory responses, followed by CNT-2 and crocidolite asbestos.

The European College of Veterinary Pathologists (ECVP): the professional body for European veterinary pathologists.

The European College of Veterinary Pathologists (ECVP) was established in 1995 with the aim of advancing veterinary pathology and promoting high standards within the specialty in Europe. The ECVP is one of 21 European colleges recognized by the European Board of Veterinary Specialisation (EBVS), which represents a quality-assurance system for European veterinary specialists. Until the ECVP was founded, there was no unified European system recognizing the specialty of pathology, and many European countries followed their own qualification systems, which varied in form and standard. The ECVP provides an annual certifying examination, the passing of which is required to gain membership (diplomate status) in the college. This qualification is now accepted on equal terms by the well-established American College of Veterinary Pathologists (ACVP). In line with EBVS requirements, the ECVP has also established a standard continuing professional development (CPD) and re-registration system for its membership. Furthermore, it has promoted and unified European post-graduate training in veterinary pathology by setting up requirements for residency training programs and making registration and monitoring of these programs by the ECVP a prerequisite for approval of an institution as a training facility. The concurrent establishment, together with the European Society of Veterinary Pathology, of an annual summer school that trains residents for the certifying examination has further fostered European post-graduate training. Within 10 years, the ECVP has succeeded in establishing common standards and a unified approach to veterinary pathology throughout Europe. This article describes the evolution and organization of the ECVP.

The ECVP/ESVP summer school in veterinary pathology: high-standard, structured training for young veterinary pathologists.

This article describes the ECVP/ESVP Summer School in Veterinary Pathology, a new annual two-week European training facility established by the European College of Veterinary Pathologists (ECVP) in collaboration with the European Society of Veterinary Pathology (ESVP). The aim of the Summer Schools is to provide Europe-wide, harmonized, top-standard theoretical and practical post-graduate training for veterinarians specializing in veterinary pathology. In particular, it aims to support trainees in veterinary pathology in their individual preparation for the ECVP certifying examination. Ultimately, it aims to provide young pathologists with the skills and knowledge necessary to participate in international, high-quality research and the tools for applying international standards to their own research and for independent study for the ECVP certifying examination, even if they do not work in comparable academic environments and do not have the same level of local support and training. The ECVP/ESVP Summer Schools take place in European countries, with local organization from a university department of veterinary pathology. Each event comprises modules provided by internationally recognized specialists in their specific fields of expertise on different organ systems, diseases of specific species, specific techniques, and specific topics relevant to pathology, forming a cycle of four events to cover all major topics. Every two years a mock exam is organized as a tool to monitor individual progress in preparing for the ECVP certifying examination.

Perinatal exposure to PCB 153, but not PCB 126, alters bone tissue composition in female goat offspring.

The aim of this study was to investigate if environmentally relevant doses of the putative estrogenic non dioxin-like PCB 153 and the dioxin-like PCB 126 caused changes in bone tissue in female goat offspring following perinatal exposure. Goat dams were orally dosed with PCB 153 in corn oil (98 microg/kg body wt/day) or PCB 126 (49 ng/kg body wt/day) from day 60 of gestation until delivery. The offspring were exposed to PCB in utero and through mother's milk. The suckling period lasted for 6 weeks. Offspring metacarpal bones were analysed using peripheral quantitative computed tomography (pQCT) after euthanisation at 9 months of age. The diaphyseal bone was analysed at a distance of 18% and 50% of the total bone length, and the metaphyseal bone at a distance of 9%. Also, biomechanical three-point bending of the bones was conducted, with the load being applied to the mid-diaphyseal pQCT measure point (50%). PCB 153 exposure significantly decreased the total cross-sectional area (125 mm(2)+/-4) versus non-exposed (142 mm(2)+/-5), decreased the marrow cavity (38 mm(2)+/-4) versus non-exposed (50 mm(2)+/-3) and decreased the moment of resistance (318 mm(3)+/-10) versus non-exposed (371 mm(3)+/-20) at the diaphyseal 18% measure point. At the metaphyseal measure point, the trabecular bone mineral density (121 mg/cm(3)+/-5) was increased versus non-exposed (111 mg/cm(3)+/-3). PCB 126 exposure did not produce any observable changes in bone tissue. The biomechanical testing of the bones did not show any significant changes in bone strength after PCB 153 or PCB 126 exposure. In conclusion, perinatal exposure to PCB 153, but not PCB 126, resulted in altered bone composition in female goat offspring.

Reduced apoptosis in sheep ileal Peyer's patch is associated with low levels of follicle centre carbonic anhydrase reactivity.

Apoptosis in lymphoid follicles of the ileal Peyer's patch (IPP) in 21 sheep of two different age groups was visualized by the TdT-mediated dUTP nick end-labelling (TUNEL) method, and quantified using computer-assisted image analysis. The IPP follicle carbonic anhydrase (CA) reactivity was evaluated in the same samples. No significant differences with respect to apoptosis and CA reactivity were found between sheep aged 5 and 11 months. Individual variation in apoptotic activity correlated with the follicle centre CA reactivity. The group of animals found to have predominantly atypical ileal lymphoid follicles (more than 80% of total number of follicles) with features resembling jejunal Peyer's patch follicles, had lower number of apoptotic cells and reduced CA reactivity compared to the rest of the animals. The differences in CA reactivity in the follicle centres probably represent a variation in the presence of CA rich approximately 50 nm membrane-bounded particles known to be a feature of the sheep IPP. The present results suggest that the particles are involved in the modulation of the lymphocyte proliferation of the IPP follicles.

NCR1+ cells appear early in GALT development of the ovine foetus and acquire a c-kit+ phenotype towards the end of gestation.

The amount, distribution and phenotype of ovine NCR1+ cells were investigated during developing GALT from day 70 of gestation. Antibodies against CD3 and CD79 were used to identify the compartments of GALT, and the localization of NCR1+ cells were correlated within these structures. Markers CD34 and c-kit, in addition to Ki67, were used to investigate possible origin and the stage of development of the NCR1+ cells. NCR1+ cells were present as single cells in the subepithelial tissue as early as 70 days of gestation, and were predominantly present in the T cell rich IFAs and domes as these intestinal wall compartments developed. While NCR1+ cells proliferated more intensively at mid-gestation (70-104 days), the number of NCR1+ cells also expressing c-kit, increased at the end of gestation. In conclusion, NCR1+ cells appeared early in T cell areas of the gut and displayed a phenotype consistent with intermediate stages of cNK cells and/or a subpopulation of ILC22.

Lymphoid follicles of different phenotype appear in ileum during involution of the sheep ileal Peyer's patch.

The ileal Peyer's patch (IPP) of young sheep is a site of diversification of the primary antibody repertoire and where involution takes place at young age. Tissue samples from the ileum were collected in 134 animals aged from 1 month to 6 years, and IPP follicle phenotypes were characterised. We describe a new type of ileal lymphoid follicles that became relatively more frequent during involution, and had numerous intrafollicular T-cells and BAQ44A+ B-cells and large interfollicular T-cell areas. As opposed to classical IPP follicles in which the BAQ44A+ cells were confined to the narrow follicle-neck region, the novel atypical ileal lymphoid follicle had these cells distributed throughout the follicle. The relative distribution of cell types in the typical IPP follicle remained fairly constant during involution. Many animals older than 9 months (64/92) still had had typical IPP follicles and even sheep 4 years and older (5/9) had IPP-type follicles.

Acute non-ambulatory tetraparesis with absence of the dens in two large breed dogs: case reports with a radiographic study of relatives.

BACKGROUND: Non-ambulatory tetraparesis with an absence of the dens of C2 (axis) has not previously been reported in large breed dogs. An absence or hypoplasia of the dens has been reported in both small, medium and large breed dogs, but not in closely related animals. METHODS: Two young large-breed dogs (a German shepherd and a Standard poodle) both with an acute onset of non-ambulatory tetraparesis were subjected to physical, neurological and radiographic examinations. Both dogs were euthanased and submitted for postmortem examination within one week of onset of clinical signs. To investigate possible heritability of dens abnormalities, oblique radiographs of the cranial cervical vertebrae were taken of nine and eighteen dogs related to the German shepherd and the Standard poodle, respectively. RESULTS: Absence of the dens, atlantoaxial instability and extensive spinal cord injury was found in both case dogs. Radiographs revealed a normal dens in both parents and in the seven littermates of the German shepherd. An absence or hypoplasia of the dens was diagnosed in six relatives of the Standard poodle. CONCLUSIONS: Atlantoaxial subluxation with cervical spinal cord injury should be considered as a differential diagnosis in non-ambulatory tetraparetic young large breed dogs. Absence of the dens and no history of external trauma increase the likelihood for this diagnosis. This study provides evidence to suggest that absence or hypoplasia of the dens is inherited in an autosomal way in Standard poodle dogs.

Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio).

Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjøsa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjøsa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjøsa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjøsa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjøsa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjøsa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.