Canine Liver Research Articles

THE FIRST ATTEMPT OF THE BIOKINETIC GA-67 MODEL APPLICATION TO CANINE LIVER CARCINOMA: CASE-CONTROL STUDY VIAIN-VIVOGAMMA CAMERA/8-SLICE CT TECHNIQUE

The biokinetic model of Ga-67 evolution was elaborated in this study for the case-control group of canine liver carcinoma via in-vivo gamma camera/8-slice CT technique. One liver carcinoma dog and two normal dogs were anesthetized with the further whole body scanning by a gamma camera to acquire the time-dependent Ga-67 concentration variations among eight compartments, namely: 1. body fluid, 2. liver, 3. GI Tract, 4. kidney, 5. heart, 6. remainder, 7. bladder, and 8. excretion. Each compartment was assumed to have a unique biological half-life and to be connected to other ones. The initial simplification of assigned compartments was performed based on the general-purpose biokinetic model recommended by the ICRP-30 report. Each object/dog underwent eight scans within 72[Formula: see text]h. The time-dependent empirical data were normalized to the maximal counts/pixel/sec and then integrated with the theoretical estimates, in order to optimize the correlations among compartments. A self-developed program run in MATLAB was used to reflect the actual performance acquired from the gamma camera scanning, while the dimensionless agreement (AT) was applied to assess the discrepancies between empirical and theoretical results. An AT of zero implies a perfect agreement between the theoretical and empirical results, while AT under 20 indicates an excellent consistency between the optimal computational and empirical data, whereas a wide fluctuation of the obtained ATs in the range of 7%–60% corresponded to a medium range of data disagreement in this study. The liver carcinoma dog has revealed a longer biological half-life than normal dogs in the limited range (40 versus 35 or 15[Formula: see text]h). However, the quantified data for other compartments and branching ratios among compartments provided a quite robust substantiation for constructing the biokinetic model of Ga-67 being administrated in the canine hepatic survey.

Melatonin prevents senescence of canine adipose-derived mesenchymal stem cells through activating NRF2 and inhibiting ER stress.

Transplantation of adipose-derived mesenchymal stem cells (ADMSCs) can aid in the treatment of numerous diseases in animals. However, natural aging during in vitro expansion of ADMSCs prior to their use in transplantation restricts their beneficial effects. Melatonin is reported to exert biorhythm regulation, anti-oxidation, and anti-senescence effects in various animal and cell models. Herein, by using a senescent canine ADMSCs (cADMSCs) cell model subjected to multiple passages in vitro, we investigated the effects of melatonin on ADMSCs senescence. We found that melatonin alleviates endoplasmic reticulum stress (ERS) and cell senescence. MT1/MT2 melatonin receptor inhibitor, luzindole, diminished the mRNA expression levels and rhythm expression amplitude of Bmal1 and Nrf2 genes. Nrf2 knockdown blocked the stimulatory effects of melatonin on endoplasmic reticulum-associated degradation (ERAD)-related gene expression and its inhibitory effects on ERS-related gene expression. At the same time, the inhibitory effects of melatonin on the NF-κB signaling pathway and senescence-associated secretory phenotype (SASP) were blocked by Nrf2 knockdown in cADMSCs. Melatonin pretreatment improved the survival of cADMSCs and enhanced the beneficial effects of cADMSCs transplantation in canine acute liver injury. These results indicate that melatonin activates Nrf2 through the MT1/MT2 receptor pathway, stimulates ERAD, inhibits NF-κB and ERS, alleviates cADMSCs senescence, and improves the efficacy of transplanted cADMSCs.

Targeted Drug and Metabolite Imaging: Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

Mass spectrometry imaging (MSI) has proven to be a valuable tool for drug and metabolite imaging in pharmaceutical toxicology studies and can reveal, for example, accumulation of drug candidates in early drug development. However, the lack of sample cleanup and chromatographic separation can hamper the analysis due to isobaric interferences. Multiple reaction monitoring (MRM) uses unique precursor ion-product ion transitions to add specificity which leads to higher selectivity. Here, we present a targeted imaging platform where desorption electrospray ionization is combined with a triple quadrupole (QqQ) system to perform MRM imaging. The platform was applied to visualize (i) lipids in mouse brain tissue sections and (ii) a drug candidate and metabolite in canine liver tissue. All QqQ modes were investigated to show the increased detection time provided by MRM as well as the possibility to perform dual polarity imaging. This is very beneficial for lipid imaging because some phospholipid classes ionize in opposite polarity (e.g., phosphatidylcholine/sphingomyelin in positive ion mode and phosphatidylserine/phosphatidylethanolamine in negative ion mode). Drug and metabolite images were obtained to show its strength in drug distribution studies. Multiple MRM transitions were used to confirm the local presence and selective detection of pharmaceutical compounds.

The value of protection device in microwave ablation at canine liver risk area

Objective To explore the application value of protective device in microwave ablation at canine liver risk area, and the role of the device in reducing complications during ablation. Methods Six healthy mongrel dogs were randomly divided into two groups: group A, used protective devices; group B, unprotected. Conventional gray-scale ultrasound and contrast-enhanced ultrasound (CEUS) were performed before treatment to identify the ablation area (the right lobe of the liver near diaphragm, 1 cm from the surface of the liver). The two groups were treated percutaneous puncture liver microwave ablation under real time ultrasound-guided with the same ablation power (50 W) and equivalent ablation time (600 s). Liver specimens were evaluated histological examination to evaluate the necrotic area of ablation and the degree of diaphragm′s injury. Results ①There was no significance difference in the ablation range between two groups[ (3.3±0.1)cm vs (3.5±0.1)cm, P=0.184]; ②The tolerance of group A was better than group B during ablation treatment; ③The mean maximum ablation diameter in group A was significantly lower than that in group B [(2.1±0.1)cm vs (5.3±0.2)cm, P=0.001]. Meanwhile the pathological results showed that the damage degree in group A was significantly lower than that in group B. Conclusions This protective device has an obvious protective effect for microwave ablation of the risk area in liver (adjacent diaphragm, hepatic envelope). Our experiment suggests that this protective can reduce the damage of adjacent organ and the complications during the ablation surgery. Key words: Microwave ablation; Liver; Diaphragm; Artificialascites; Dogs

Abstract 4918: Pharmacokinetics and toxicity studies of AT1965, a B-cell activating immunotherapy

Abstract Introduction: Cancer immunotherapy is the emerging paradigm in the search for a cure for cancer. AT1965 is a novel therapeutic that demonstrates remarkable anti-tumor activity across different preclinical tumor models, mediated through the activation of B-cells. In this study, we have evaluated the metabolism, pharmacokinetics (PK) and toxicity of AT1965 in rodent and non-rodent species. Methods: AT1965 metabolism was studied in rodent, canine and primate liver microsomes. The PK of AT1965 was determined in 4T1 tumor bearing female Balb/c mice; female Sprague Dawley rats and female Beagle dogs at a variety of doses and schedules following single intravenous (i.v) injections. The extent and duration of the exposure was monitored over 120 hours in mice and rats, and till 192 hours in dogs, post dosing. Concentrations of AT1965 in plasma samples were determined either by LC-MS/MS or Atomic Absorption Spectroscopy. The toxicity of AT1965 in male and female Sprague Dawley Rats following single dose i.v. administration of 43.7, 87.4 and 174.8 mg/kg dosage of AT1965 were examined. During toxicity studies, clinical health status was observed daily for 14 days after treatment, followed by necropsy. Results: AT1965 metabolism evaluated in mouse, rat, dog, monkey and human liver microsomes indicated high stability across all species. PK studies reveal that AT1965 exhibited a high exposure, slow systemic clearance and a proportional increase in dose normalized exposure across species. The elimination half-life (T1/2) varied from 34-40 hours in tumor-bearing mice to 29-44 hours in rats and ~ 66 hours in dogs. The excretion of AT1965 was predominantly through hepatobiliary route, with minimal renal clearance. Single i.v. administered acute dose toxicity studies for AT1965 reveal 87.4 mg/kg and 174.8 mg/kg as MTD and toxic doses respectively in rats. Conclusion: The excellent pharmacokinetic profile of AT1965 showing high exposure and enhanced elimination half-life tested across species, in addition to striking antitumor activity coupled with low toxicity in multiple rodent tumor models, provide a compelling rationale for the development of AT1965 as a novel immunotherapeutic agent in the clinic. Citation Format: Aniruddha Sengupta, Mallik Samarla, Manoj Pandey, Arindam Sarkar, Monideepa Roy, Shiladitya Sengupta. Pharmacokinetics and toxicity studies of AT1965, a B-cell activating immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4918.

Organ‑specific expression of the divalent ion channel proteins NCKX3, TRPV2, CTR1, ATP7A, IREG1 and HEPH in various canine organs.

Transmembrane cation channels include those for calcium, copper and iron ion transport. Each channel has physiological significance, and all have been associated with disease. However, the comparative study of transcriptional‑translational levels in canine organs has not been previously reported. In the present study, organ‑specific expression of calcium channels, including sodium/potassium/calcium exchanger 3 (NCKX3) and transient receptor potential cation channel subfamilyV member 2 (TRPV2), copper channels, including high affinity copper uptake protein 1 (CTR1) and copper‑transporting ATPase 1 (ATP7A), and iron channels, including iron‑regulated transporter 1 (IREG1) and hephaestin (HEPH) proteins and their mRNAs were examined in the canine duodenum, kidney, spleen and liver. NCKX3 protein expression was highest in the kidney, moderate in the duodenum, and lowest in the spleen and liver, whereas TRPV2 protein was highly expressed in the kidney, duodenum and liver, and was low in the spleen. The CTR1 protein expression level was highest in the liver, followed (in descending order) by the duodenum, kidney and spleen. The ATP7A protein expression level was highest in the duodenum and lowest in the spleen. The IREG1 protein expression level was highest in the liver, followed (in descending order) by the kidney, duodenum and spleen. The HEPH protein level was high in liver, moderate in the duodenum and kidney, and low in the spleen. The results of the immunohistochemistry analysis demonstrated ion channel protein localizations. These results suggested that cation channel proteins are differentially expressed among canine organs, and they may be involved in organ‑specific functions associated with the maintenance of physiological homeostasis.

What is your diagnosis? Generalized nodular change in a canine liver.

What is your diagnosis? Generalized nodular change in a canine liver.

Enantioselective capillary electrophoresis provides insight into the phase II metabolism of ketamine and its metabolites in vivo and in vitro.

Glucuronidation catalyzed by uridine-5'-diphospho-glucuronosyl-transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O-glucuronidation is more common than N-glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β-glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Concentrations of HNK diastereomers and norketamine were significantly higher in comparison to untreated urine and an increase of ketamine and DHNK levels was found in selected but not all samples. This suggests that O-glucuronides of HNK and N-glucuronides of the other compounds are formed in equines. N-glucuronidation of norketamine was studied in vitro with liver microsomes of different species and the single human enzyme UGT1A4. With equine liver microsomes (ELM) a stereoselective N-glucuronidation of norketamine was found that compares well to the results obtained with urines collected after ketamine administration. No reaction was observed with canine liver microsomes, human liver microsomes and UGT1A4. Incubation of ketamine and DHNK with ELM did not reveal any glucuronidation. Enantioselective CE is suitable to provide insight into the phase II metabolism of ketamine and its metabolites.

Comparison of predicted intrinsic hepatic clearance of 30 pharmaceuticals in canine and feline liver microsomes

1. Known cytochrome P450 (CYP) substrates in humans are used in veterinary medicine, with limited knowledge of the similarity or variation in CYP metabolism. Comparison of canine and feline CYP metabolism via liver microsomes report that human CYP probes and inhibitors demonstrate differing rates of intrinsic clearance (CLint).2. The purpose of this study was to utilize a high-throughput liver microsome substrate depletion assay, combined with microsomal and plasma protein binding to compare the predicted hepatic clearance (CLhep) of thirty therapeutic agents used off-label in canines and felines, using both the well-stirred and parallel tube models.3. In canine liver microsomes, 3/30 substrates did not have quantifiable CLint, while midazolam and amitriptyline CLint was too rapid for accurate determination. A CLhep was calculated for 29/30 substrates in feline microsomes. Overall, canine CLhep was faster compared to the feline, with fold differences ranging from 2–20-fold.4. A comparison between the well-stirred and parallel tube model indicates that the parallel tube model reports a slighter higher CLhep in both species.5. The differences in CYP metabolism between canine and feline highlight the need for additional research into CYP expression and specificity.

Comparison for Radiographic Measurements of Canine Liver Size by Left and Right Recumbency

Comparison for Radiographic Measurements of Canine Liver Size by Left and Right Recumbency

Comparison of diagnostic accuracy of laparoscopic 3 mm and 5 mm cup biopsies to wedge biopsies of canine livers.

BackgroundDiagnostic accuracy of the 3 mm laparoscopic cup biopsy forceps for collection of tissue samples from canine livers is unproven.Hypotheses/ObjectivesCompare sample surface area and portal triad count between 3 mm and 5 mm laparoscopic cup biopsies and compare the histologic diagnosis obtained by each instrument to a standard necropsy wedge. The hypothesis was that more portal triads and greater sample surface area would be found with the 5 mm samples and the laparoscopic instruments would not have significantly different levels of agreement with necropsy wedge diagnosis.AnimalsTwenty‐one client‐owned dogs undergoing necropsy.MethodsProspective ex vivo study. Three samples (3 mm, 5 mm, and wedge) were taken of 2 different hepatic divisions within 24 hours of death. Morphologic diagnosis, World Small Animal Veterinary Association histologic features, surface area, and portal triad numbers were compared among the 3 samples.ResultsThere were significantly more portal triads (mean 21.4 versus 13.8; P < .0001) and a higher surface area (20.3 mm2 versus 11.5 mm2; P < .0001) in the 5 mm samples compared to 3 mm samples. Kappa coefficients and percent agreement for histologic diagnosis as compared to the wedge biopsy were not significantly different between the 2 instrument sizes (κ = 0.383 and 0.436, respectively; 67% and 69%, respectively).Conclusions and Clinical ImportanceDespite yielding smaller sample sizes, the 3 mm laparoscopic cup biopsy has a similar level of histologic diagnostic accuracy to the 5 mm instrument.

A single-nucleotide polymorphism in the canine cytochrome b5 reductase (CYB5R3) gene is associated with sulfonamide hypersensitivity and is overrepresented in Doberman Pinschers.

Canine sulfonamide hypersensitivity (HS) has been associated with a variant in the cytochrome b5 reductase gene (CYB5R3 729A>G), which encodes a drug-detoxifying enzyme. Study objectives were to determine variant allele frequency in Doberman Pinschers (DOBE), a breed which may be predisposed to sulfonamide HS, and to characterize the effects of CYB5R3 729G on gene expression and function. CYB5R3 729A>G allele frequencies were compared between DOBE (n=24) vs. non-Doberman (non-DOBE; n=60) dogs. CYB5R3mRNA expression, protein expression, and reduction of sulfamethoxazole hydroxylamine were compared between banked canine liver samples of 729AA vs. GG genotype. The 729G allele was overrepresented in DOBE (1.00) vs. non-DOBE dogs (0.567, p<.0001). mRNA and protein expressions as well as cyt b5 reductase activity were similar between livers of AA and GG genotype. All Doberman Pinschers in this study were homozygous for CYB5R3 729G, which could contribute to this breed's apparent predisposition to sulfonamide HS. However, CYB5R3 729G does not alter sulfamethoxazole detoxification capacity, so a direct role could not be demonstrated. It is possible that this marker is linked to another contributing variant.

Non-invasive quantification of hepatic fat content in healthy dogs by using proton magnetic resonance spectroscopy and dual gradient echo magnetic resonance imaging.

The objective of the present study was to describe two non-invasive methods for fat quantification in normal canine liver by using magnetic resonance imaging (MRI) and spectroscopy. Eleven adult beagle dogs were anesthetized and underwent magnetic resonance examination of the cranial abdomen by performing morphologic, modified Dixon (mDixon) dual gradient echo sequence, and proton magnetic resonance spectroscopy (1H MRS) imaging. In addition, ultrasonographic liver examination was performed, fine-needle liver aspirates and liver biopsies were obtained, and hepatic triglyceride content was assayed. Ultrasonographic, cytologic, and histologic examination results were unremarkable in all cases. The median hepatic fat fraction calculated was 2.1% (range, 1.3%–5.5%) using mDixon, 0.3% (range, 0.1%–1.0%) using 1H MRS, and 1.6% (range 1.0%–2.5%) based on triglyceride content. The hepatic fat fractions calculated using mDixon and 1H MRS imaging were highly correlated to that based on triglyceride content. A weak correlation between mDixon and 1H MRS imaging was detected. The results show that hepatic fat content can be estimated using non-invasive techniques (mDixon or 1H MRS) in healthy dogs. Further studies are warranted to evaluate the use of these techniques in dogs with varying hepatic fat content and different hepatic disorders.

Hyaluronic acid and TGF-β1 in dogs with hepatobiliary diseases

The aim of the study was to assess serum hyaluronic acid (HA) and transforming growth factor beta 1 (TGF-β1) concentrations: 1) to differentiate hepatic fibrosis from other forms of liver disease, and 2) for the non-invasive staging of canine liver fibrosis. We also evaluated the association between serum HA concentration and the size of the shunt vessel as an indirect marker of decreased liver clearance in patients with single congenital vascular anomaly. Forty-one healthy client-owned dogs and forty dogs diagnosed with hepatobiliary disease were enrolled in the prospective study. Patients were divided into 4 subgroups: 1) congenital portosystemic shunts (CPSS); 2) parenchymal diseases (a. mild and moderate fibrosis, b. advanced fibrosis and cirrhosis); 3) hepatic neoplasia; 4) biliary tract disorders based on thorough clinical, ultrasound and histopathological examination. Serum HA and TGF-β1 concentrations were measured using ELISA. The HA concentration was significantly increased in patients with advanced liver fibrosis/cirrhosis (P &lt; 0.001) and CPSS (P &lt; 0.001) compared to healthy dogs. Using a cut-off HA concentration of 135.94 ng/ml, the sensitivity and specificity for diagnosis for advanced liver fibrosis/cirrhosis was 100% (95% CI, 50.6–100) and 90.8% (95% CI, 81.6–95.7), respectively. The TGF-β1 levels did not significantly differ among groups (P = 0.180). Negligible correlation was found between serum HA concentration and the size of portosystemic shunt vessel (rs = 0.07; P = 0.831). These findings suggest that serum HA concentration is a potential non-invasive biomarker for advanced liver fibrosis and/or cirrhosis in dogs. The utility of measuring serum concentration of TGF-β1 for diagnosing canine liver fibrosis was not supported.

Stellate cells in livers of dogs with portal vein hypoperfusion

Hepatic stellate cells play a crucial role in the development of liver fibrosis. In a damaged liver, stellate cells undergo activation, which is manifested as a change of their phenotype: differentiation of stellate cells to myofibroblast-type cells, expression of alpha-Smooth Muscle Actin, their proliferation and a reduction in the size of cytoplasmic lipid droplets. The aim of this study was to determine the number and morphology of stellate cells in the canine liver affected by congenital portosystemic shunt (PSS) and portal vein hypoplasia – hepatic microvascular dysplasia (PVH-HMD). The material for investigation were archived paraffin blocks with liver samples collected supravitally from six dogs with PSS, six dogs with PVH-HMD and six healthy dogs. On the HE-stained sections, the number of stellate cells per 100 hepatocytes was counted (Sztark method) and the diameter of veins in the hepatic triads was measured (light microscope Olympus BX 43, SC30 camera, CellSens Entry 2011 Olympus). In addition, the diameter of lipid droplets in stellate cells was measured (computed image analysis system LUCIA 4.21). The results were analysed statistically (the Kruskal-Wallis test followed by Dunn’s post-hoc procedure; significance level (α) at 0.05; Statistica 12 StatSoft Inc.). The degree of liver fibrosis was determined (Masson’s method of slide stain; Scheuer scale). The liver samples from the dogs with PSS and PVH-HMD were stained immunohistochemically with Monoclonal Mouse Anti-Human Smooth Muscle Actin (α-SMA), clone 1A4, antibodies (DAKO). Portosystemic shunt and primary portal vein hypoplasia in the dog results in a reduction in the diameter of portal vein branches and in insufficient portal blood flow through the liver. In the material investigated, this was particularly evident in the animals affected by PSS: such dogs had a significantly smaller diameter of the veins than did the healthy dogs (p&lt;0.001) or the dogs with PVH-HMD (p=0.023). Fibrosis and the expression of α-SMA were stronger in the dogs with PSS than in those with PVH-HMD. Moreover, the dogs with PSS had a significantly higher average number of stellate cells than the healthy animals (p=0.007) did. However, the examination of the material revealed an enlargement of cytoplasmic lipid droplets: the dogs with PSS had a significantly larger diameter of lipid vacuoles in the cytoplasm of stellate cells than did the healthy animals (p&lt;0.001) or the dogs with PVH-HMD (p=0.043); the dogs with PVH-HMD had lipid droplets with a significantly larger diameter than the healthy animals (p&lt;0.001) did. Hypoperfusion of the liver and the accompanying regressive lesions in hepatocytes result mainly in an increased number of stellate cells and stronger expression of α-SMA, while cytoplasmic lipid droplets in the stellate cells are not reduced in size. The present study indicates the need for detailed analyses of clinical cases and warrants further comprehensive studies of comparative hepatopathology because it demonstrates differences between humans and dogs in the morphological indicators of hepatic stellate cell activation in chronic liver damage.

Clinical relevance of radiographic linear branching mineral opacities in the canine liver.

To assess the prevalence, clinical significance and breed distribution of linear branching mineralisation superimposed on the hepatic radiographic silhouette in dogs. Retrospective review of radiographs or ultrasound images of dogs showing branching mineralisation in the liver. Over the 30-year review period, 17 cases were identified and the mineralisation had a predominantly ventral distribution. Seven of the 17 were cavalier King Charles spaniels, and four of the total 17 dogs were diagnosed with hepatobiliary system disease. Five dogs had repeat radiographs, of which four showed no change in the pattern and one developed the pattern 6 years after being diagnosed with cholangiohepatitis. Serum calcium concentrations were normal in all patients. Liver enzymes were markedly elevated only in the dog diagnosed with cholangiohepatitis. Histology performed on three patients showed no convincing evidence of primary liver disease. Branching mineralisation in the liver parenchyma is a rare finding in dogs with little or no clinical significance and cavalier King Charles spaniels may be predisposed. Biopsy of the liver of affected dogs with no clinical or clinicopathological evidence of liver disease is unlikely to be helpful in these cases.

Characterization of a low expression haplotype in canine glutathione S-transferase (GSTT1) and its prevalence in golden retrievers.

Glutathione S-transferase-theta (GSTT1) is a carcinogen detoxification enzyme, and low activity variants are associated with lymphoma in humans. We recently found a variant in the 3' untranslated region (UTR) of canine GSTT1, *101_102insT, which was predicted to change miRNA binding and was found in 5 of 17 golden retriever (GR) dogs with lymphoma but none of 14 healthy GRs. The aim of this study was to determine whether this variant led to decreased GSTT1 expression and was a discernible risk factor for lymphoma within the GR breed. On resequencing, *101_102insT appeared to be in complete linkage disequilibrium with 3 additional 3'UTR variants, leading to the inferred haplotype *3T>C; *101_102insT; *190C>A; *203T>C. In canine livers that were heterozygous for this variant haplotype, GSTT1 protein expression was significantly lower compared to the reference haplotype (densitometry .40 vs .64, P = .022), and GSTT1 transcript levels by qPCR were also significantly lower (fold difference .52, P = .012), without evidence of substantial allelic expression imbalance. The variant haplotype led to >50% decrease in expression in vitro (.31 ± .07 vs .64 ± .19; P = .019). We found no significant difference in minor allele frequencies between 71 GR dogs with lymphoma (MAF .162) and 33 healthy age-matched controls (MAF .136, P = .69). Our results indicate that the variant GSTT1 3'UTR haplotype containing *101_102insT reduces gene expression, which could lead to impaired carcinogen detoxification, but was not a detectable risk factor for lymphoma in GR dogs.

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate.

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) was investigated, with ATP and Na2 SO4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17β-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile.

A canine liver fibrosis model to develop a therapy for liver cirrhosis using cultured bone marrow-derived cells.

We have been developing a therapy for liver cirrhosis using cultured autologous bone marrow–derived mesenchymal stem cells (BMSCs). Before human clinical trials can be considered, the safety and efficacy of BMSC infusion in medium to large animals must be confirmed; thus, we developed a canine liver fibrosis model. A small amount of bone marrow fluid was aspirated from the canine humerus to assess the characteristics of BMSCs. We implanted a venous catheter in the stomach and a subcutaneous infusion port in the back of the neck of each canine. Repeated injection of CCl4 through the catheter was performed to induce liver cirrhosis. After 10 weeks of CCl4 injection, eight canines were equally divided into two groups: no cell infusion (control group) and autologous BMSC infusion through the peripheral vein (BMSC group). A variety of assays were carried out before and 4 weeks after the infusion. The area of liver fibrosis stained with sirius red was significantly reduced in the BMSC group 4 weeks after BMSC infusion, consistent with a significantly shortened half‐life of indocyanine green and improved liver function. Conclusion: We established a useful canine liver fibrosis model and confirmed that cultured autologous BMSC infusion improved liver fibrosis without adverse effects. (Hepatology Communications 2017;1:691–703)

Transcatheter arterial embolization in normal canine liver.

To determine the effects of selective transcatheter arterial embolization (TAE) in the normal canine liver. Experimental study. Adult Beagle dogs (n = 5). Gelatin sponge particles (GSPs) were injected through a microcatheter for selective embolization of the left hepatic artery in normal dogs. Computed tomography (CT) and histology were performed during an 8-week observation period; biochemical analysis data were obtained during a 12-week observation period after TAE. Embolization was successful in all dogs and did not induce any change in the clinical appearance of dogs. Postoperative CT was consistent with recanalization of the artery within 2 weeks of embolization in all dogs. Hepatic enzyme levels increased temporarily after embolization but gradually returned to normal ranges. Histological examinations did not differ between treated and untreated liver tissues. TAE appears safe in normal dogs observed for 12 weeks. Arterial recanalization seems to occur within 2 weeks after injection of GSPs in the left hepatic artery. Selective TAE of the hepatic artery was well tolerated in normal dogs. Selective TAE may be applicable to canine hepatocellular carcinoma.