Comparative hepatotoxicity of novel lithium bis(trifluoromethanesulfonyl)imide (LiTFSI, ie. HQ-115) and legacy Perfluorooctanoic acid (PFOA) in male mice: Insights into epigenetic mechanisms and pathway-specific responses.

Lithium Bis(trifluoromethanesulfonyl)imide (LiTFSI ie. HQ-115), a polymer electrolyte used in energy applications, has been detected in the environment, yet its health risks and environmental epigenetic effects remain unknown. This study aims to unravel the potential health risks associated with LiTFSI, investigate the role of DNA methylation-induced toxic mechanisms in its effects, and compare its hepatotoxic impact with the well-studied Perfluorooctanoic Acid (PFOA). Using a murine model, six-week-old male CD1 mice were exposed to 10 and 20 mg/kg/day of each chemical for 14 days as 14-day exposure and 1 and 5 mg/kg/day for 30 days as 30-day exposure. Results indicate that PFOA exposure induced significant hepatotoxicity, characterized by liver enlargement, and elevated serum biomarkers. In contrast, LiTFSI exposure showed lower hepatotoxicity, accompanied by mild liver injuries. Despite higher bioaccumulation of PFOA in serum, LiTFSI exhibited a similar range of liver concentrations compared to PFOA. Reduced Representative Bisulfite Sequencing (RRBS) analysis revealed distinct DNA methylation patterns between 14-day and 30-day exposure for the two compounds. Both LiTFSI and PFOA implicated liver inflammatory pathways and lipid metabolism. Transcriptional results showed that differentially methylated regions in both exposures are enriched with cancer/disease-related motifs. Furthermore, Peroxisome proliferator-activated receptor alpha (PPARα), a regulator of lipid metabolism, was upregulated in both exposures, with downstream genes indicating potential oxidative damages. Overall, LiTFSI exhibits distinct hepatotoxicity profiles, emphasizing the need for comprehensive assessment of emerging PFAS compounds.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Chronic Inflammatory Enteropathy and Low-Grade Intestinal T-Cell Lymphoma Are Associated with Altered Microbial Tryptophan Catabolism in Cats.

Chronic inflammatory enteropathy (CIE) and low-grade intestinal T-cell lymphoma (LGITL) are common chronic enteropathies (CE) in cats. Enteric microbiota dysbiosis is implicated in the pathogenesis of CE; however, the mechanisms of host-microbiome interactions are poorly understood in cats. Microbial indole catabolites of tryptophan (MICT) are gut bacterial catabolites of tryptophan that are hypothesized to regulate intestinal inflammation and mucosal barrier function. MICTs are decreased in the sera of humans with inflammatory bowel disease and previous studies identified altered tryptophan metabolism in cats with CE. We sought to determine whether MICTs were decreased in cats with CE using archived serum samples from cats with CIE (n = 44) or LGITL (n = 31) and healthy controls (n = 26). Quantitative LC-MS/MS was used to measure serum concentrations of tryptophan, its endogenous catabolites (kynurenine, kynurenate, serotonin) and MICTs (indolepyruvate, indolealdehyde, indoleacrylate, indoleacetamide, indoleacetate, indolelactate, indolepropionate, tryptamine). Serum concentrations of tryptophan, indolepropionate, indoleacrylate, indolealdehyde, indolepyruvate, indolelactate were significantly decreased in the CIE and LGITL groups compared to those in healthy controls. Indolelactate concentrations were significantly lower in cats with LGITL compared to CIE (p = 0.006). Significant correlations were detected among serum MICTs and cobalamin, folate, fPLI, and fTLI. Our findings suggest that MICTs are promising biomarkers to investigate the role of gut bacteria in the pathobiology of chronic enteropathies in cats.

Propylparaben inhibits mouse cultured antral follicle growth, alters steroidogenesis, and upregulates levels of cell-cycle and apoptosis regulators.

Propylparaben is prevalently used in cosmetics, pharmaceuticals, and foods; yet, its direct effects on the mammalian ovary are unknown. We investigated the direct effects of propylparaben on the growth and steroidogenic function of mouse antral follicles. Antral follicles were isolated from the ovaries of Swiss mice (age: 32-42 days) and cultured in media with dimethylsulfoxide vehicle control or propylparaben (0.01-100 µg/mL) for 24-72 h. Follicle diameter was measured every 24 h to assess growth. Follicles and media were collected at 24 and 72 h for gene expression and hormone measurements. Propylparaben (100 µg/mL) significantly inhibited follicle growth (48-72 h). Further, propylparaben exposure increased expression of cell cycle regulators (Cdk4, Cdkn1a), an apoptotic factor (Bax), and a key steroidogenic regulator (Star). In media, propylparaben decreased accumulation of dehydroepiandrosterone-sulfate, but increased testosterone and 17ß-estradiol. Overall, our findings suggest that propylparaben disrupts antral follicle growth and steroidogenic function by altering the cell-cycle, apoptosis, and steroidogenesis pathways.

Dermoid sinus type VI associated with spina bifida and tethered cord syndrome in a French Bulldog.

A 4-mo-old French bulldog was presented with acute onset pain and reluctance to move. A tubular structure arising in the dorsal thoracic midline and extending from a cutaneous orifice into deeper tissues was palpated on physical examination. Computed tomography with sinography revealed a dermoid sinus associated with spina bifida at the level of T3-T4. On surgical exploration, the dermoid sinus was found to communicate with the dura. Histology confirmed the diagnosis and classification as a type VI dermoid sinus. The pain response and hyperesthesia were suspected to be the result of tethered cord syndrome. Complete resolution of clinical signs was appreciated post-surgery, with the patient still free of clinical signs 3 mo later.

Carcinoma in situ within an area of Barrett esophagus in a dog with megaesophagus.

A 10-y-old Irish Setter was presented with a history of recurrent episodes of regurgitation and vomiting, with more recent development of tachypnea. Megaesophagus had been diagnosed in the dog 2 y prior to this presentation. A solitary polypoid mass present immediately rostral to the lower esophageal sphincter was biopsied during percutaneous endoscopic gastrostomy tube placement. Barrett esophagus was diagnosed based on the observation of a polypoid mass with intestinal metaplasia that arose from the surrounding esophagus. Histology of the polypoid mass demonstrated squamous-to-columnar metaplasia, hyperplasia, dysplasia, and carcinoma in situ.

The Site of Bone Marrow Acquisition Affects the Myeloid to Erythroid Ratio in Apparently Healthy Dogs.

Bone marrow (BM) cytology and histopathology are complementary tools used to investigate hematological diseases. The purpose of this study was to determine if there are site-dependent differences in the diagnostic quality, myeloid to erythroid ratio (MER), and discordant findings in samples from different sites in the same dog. Eighteen apparently healthy dogs were used in the study. The sequence of sample acquisition was randomized according to a Latin square, and samples for BM cytology and histology were collected from both humeri and both ilial crests immediately after death. Board-certified clinical and anatomical pathologists read the cytology and histology, respectively. The data were analyzed using a mixed-effect model. The site of BM acquisition did not affect BM sample quality. The rate of discordant clinical findings between sites was 0.05 (95% confidence interval, 0.01-0.13). In general, by cytology, the MERs were slightly but significantly greater in samples from the ilial crests than from the humeri ( P = .01). The measured MER for histology was nearly twice that for cytology for all sites ( P < .001). In conclusion, there was a low-rate, site-dependent discordance in diagnostic findings in BM samples and differences in MER between the ilial crest and the humerus. A similar study is justified in sick dogs with hematological disease to determine the effect of sampling site on discordant findings between sites.

Proposed expansion margins for planning organ at risk volume for lenses during radiation therapy of the nasal cavity in dogs and cats.

Radiation therapy protocols for the feline or canine nasal cavity can damage epithelial cells of the posterior pole of the lens and lead to the development of cataracts. Aims of this retrospective, descriptive study were to calculate movements of the lens during radiation therapy of the nasal cavity in a sample of cats and dogs, and to propose species-specific expansion margins for planning organ at risk volume (PRV) to minimize radiation doses to the lens. All included patients were immobilized with an indexed bite block and positioned in a vacuum positioning cushion for head irradiation. On-board cone beam CT (CBCT) imaging was used for patient alignment. Both ocular lenses were contoured on the therapeutic CBCTs. Coregistration (fusion) between the planning CT and CBCTs was used to measure the movements of the lens. Two measurements were made: the differences between the centroid point of each lens as well as the displacement of the coregistrations. A total of 496 different observations were recorded from 14 cats and 52 dogs. Using the displacement results, we calculated how often the lens would be within the lens-PRV contour. We proposed that an optimal expansion margin from the lens volume of 2 mm in cats and 3 mm in dogs may be necessary in generating PRV expansion for the lens. From our results, we expect the lens would therefore be within these proposed PRV expansions in 92% of the feline measurements and 95% of the canine measurements.

Loss of Fertility in the Absence of Progesterone Receptor Expression in Kisspeptin Neurons of Female Mice.

Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the Pgr gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the Pgr deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.

Generation and characterization of an endothelin-2 iCre mouse.

A novel transgenic mouse line that expresses codon-improved Cre recombinase (iCre) under regulation of the Endothelin-2 gene (edn2) promoter was developed for the conditional deletion of genes in Endothelin-2 lineage cells and for the spatial and temporal localization of Endothelin-2 expression. Endothelin-2 (EDN2, ET-2, previously VIC) is a transcriptionally regulated 21 amino acid peptide implicated in vascular homeostasis, and more recently in female reproduction, gastrointestinal function, immunology, and cancer pathogenesis that acts through membrane receptors and G-protein signaling. A cassette (edn2-iCre) was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker in front of the endogenous start codon of the edn2 gene in a mouse genome BAC clone. The cassette was introduced into the C57BL/6 genome by pronuclear injection, and two lines of edn2-iCre positive mice were produced. The edn2-iCre mice were bred with ROSA26-lacZ and Ai9 reporter mice to visualize areas of functional iCre expression. Strong expression was seen in the periovulatory ovary, stomach and small intestine, and colon. Uniquely, we report punctate expression in the corneal epithelium, the liver, the lung, the pituitary, the uterus, and the heart. In the embryo, expression is localized in developing hair follicles and the dermis. Therefore, edn2-iCre mice will serve as a novel line for conditional gene deletion in these tissues.

Comparison between survey radiography, B-mode ultrasonography, contrast-enhanced ultrasonography and contrast-enhanced multi-detector computed tomography findings in dogs with acute abdominal signs.

Contrast-enhanced multi-detector computed tomography (CE-MDCT) is used routinely in evaluating human patients with acute abdominal symptoms. Contrast-enhanced ultrasound (CEUS) continues to be in its infancy as it relates to evaluation of the acute abdomen. The purpose of this study was to compare survey radiography, B-mode ultrasound, CEUS, and CE-MDCT findings in canine patients presenting with acute abdominal signs; with a focus on the ability to differentiate surgical from non-surgical conditions. Nineteen dogs were prospectively enrolled. Inclusion required a clinical diagnosis of acute abdominal signs and confirmed surgical or non-surgical causes for the clinical signs. Agreement for the majority of recorded imaging features was at least moderate. There was poor agreement in the identification of pneumoperitoneum and in the comparison of pancreatic lesion dimensions for B-mode vs. CEUS. The CT feature of fat stranding was detected in cases including, but not limited to, gastric neoplasia with perforation, pancreatitis, and small intestinal foreign body. Ultrasound underestimated the size and number of specific lesions when compared with CE-MDCT. Contrast-enhanced ultrasound was successful in detecting bowel and pancreatic perfusion deficits that CE-MDCT failed to identify. Accuracy for differentiation of surgical vs. non-surgical conditions was high for all modalities; 100%, 94%, and 94% for CE-MDCT, ultrasonography and survey radiography respectively. Findings indicated that CE-MDCT is an accurate screening test for differentiating surgical from non-surgical acute abdominal conditions in dogs. Focused CEUS following CE-MDCT or B-mode ultrasonography may be beneficial for identifying potentially significant hypoperfused lesions.

Congenital adenohypophyseal hypoplasia associated with secondary hypothyroidism in a 2-week-old Portuguese water dog.

This report describes the histomorphological changes of central hypothyroidism (pituitary dependent) in several target organs of thyroid hormones of a Portuguese water dog, and contrasts those with the reported features of central hypothyroidism in German shepherd dogs, in which central hypothyroidism is a part of a combined pituitary hormonal deficiency.

An unusual clinical presentation of a dog with gastrinoma.

Gastrinoma is a rare malignant neuroendocrine neoplasia that results in autonomous gastrin secretion that stimulates hypersecretion of gastric acid, resulting in severe gastric and proximal small intestinal ulcerations. The principal clinical manifestation of gastrinoma is persistent vomiting. This report describes an uncommon manifestation of pancreatic gastrinoma in a dog.

Detection of Ehrlichia canis by PCR in different tissues obtained during necropsy from dogs surveyed for naturally occurring canine monocytic ehrlichiosis.

A molecular study for the detection of Ehrlichia canis was carried out on tissues obtained at necropsy from randomly selected dogs with the intention of investigating naturally-occurring canine ehrlichiosis. The tissues evaluated for the presence of E. canis included lymph nodes, spleen, liver, bone marrow, and blood. Eight of the 18 dogs included were found to be positive for E. canis by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. Two dogs were positive for Anaplasma platys of which one dog was co-infected with E. canis and A. platys. Blood (5/8) and lymph nodes (5/8) were the tissues found to yield the highest number of positive E. canis PCR results with 7/8 dogs positive in the blood or lymph node. E. canis and A. platys DNA could be amplified by PCR when tissue samples were obtained 72h after the time of death.

Aortic thromboembolism associated with Spirocerca lupi infection.

A 2-year-old male castrated Cavalier King Charles Spaniel was presented with paraplegia, cold caudal extremities and lack of femoral pulses. A 2cm long thrombus occluding the aortic trifurcation and a 3cm long abdominal aortic aneurysm with a thrombus were detected by ultrasonographic examination. The clinical and ultrasonographic findings were consistent with aortic thromboembolism. Anti-thrombotic and vasodilative therapy was not helpful and the dog was euthanized 3 days after the onset of paraplegia. A thrombus in the aortic trifurcation, multiple thoracic and abdominal aneurysms and a distal mediastinal esophageal granuloma containing Spirocera lupi worms were found on necropsy. The abdominal aortic aneurysms formed by S. lupi larval migration are believed to be responsible for the formation of the thrombus that occluded the aortic trifurcation. This is the first report of aortic thromboembolism associated with S. lupi infection.