Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl on gene expression in the ovaries of immature Sprague-Dawley rats

We have previously shown that 3,3',4,4',5-pentachlorobiphenyl (PCB-126) vertically transferred from dams potentially exerts a direct effect on the ovaries of offspring and adversely affects female puberty. To investigate its toxicological targets in ovarian tissues, mRNAs encoding representative peptides that regulate follicular development in granulosa cells, theca cells, and oocytes were quantified using ovaries collected on postnatal days (PND) 5, 15, and 24 from the offspring of dams administered oral doses of 0, 1 or 3 microg/kg PCB-126 starting 2 weeks prior to mating and continuing until 20 days after delivery. Quantification using the real-time RT-PCR method revealed that PCB-126 lowered the amounts of mRNAs that encoded the inhibin alpha- and inhibin/activin beta A-subunits from PND 15 onwards; the amounts of mRNAs for inhibin/activin beta B-subunit, follicle-stimulating hormone (FSH) receptor, and aromatase on PND 15; and the amounts of luteinizing hormone receptor mRNA on PND 24 compared with those of the age-matched controls. In contrast, no differences were noted for mRNAs encoding c-kit, growth differentiation factor-9, bone morphogenetic protein-15, or kit ligand for any of the age groups examined. The serum FSH level on PND 24 was higher than that in the control. Since the earliest effects on the mRNAs in the rat ovaries were observed in those expressed in the granulosa cells of the growing follicles after the antral follicles had developed, molecules in granulosa cells but not in oocytes during the early stages of the antral follicles might be the primary targets of vertically transferred PCB-126.     

Influence of polychlorinated biphenyls on the secretion of oxytocin from bovine luteal cells and from granulosa cells obtained from the follicles of different size

Effect of polychlorinated biphenyles (PCBs) on viability and secretory function of luteal and granulosa cells from mature cows was studied. Luteal cells from corpora lutea of different developmental stages and granulosa cells from follicles of >1 cm< in diameter were used. Neither individual congeners (PCB-126, -77, -153) nor mixture of PCBs Aroclor Ar) 1248 at the dose of 1, 10 or 100 ng/ml affected the viability of cells (P>0.05) compared to control after 72 h of incubation. PCBs markedly increased (P<0.05-0.001) oxytocin (OT) secretion from granulosa cells. This effect was the most evident when granulosa cells from follicles <1 cm diameter was treated with PCB-77 which is assumed to stimulate both arylhydrocarbon receptor (AhR) and estradiol (E2) receptor. Even the lowest dose of this compound (1 ng/ml) outranged the effect produced by cortisol (10(-5)M) used as positive control. There was marked effect (P<0.05-0.001) of PCBs on luteal cells from days 6-15 of the estrous cycle. However, influence of PCBs on OT secretion from luteal cells on day 1-5 and 16-18 of the estrous cycle was less evident. Again, PCB-77 was the most efficient stimulator of OT secretion. While the lowest effect was found after treatment of cells with PCB-126 which has dioxin-like properties. It can be assumed that diverse effect of PCBs on female reproduction largely results from the influence of these compounds on ovarian OT secretion. Since both synthesis and secretion of ovarian OT in bovine do not markedly depend on estradiol, some alternative cellular pathways of PCBs on ovary function are suggested.     

The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the ABCG2-specific inhibitor fumitremorgin C (FTC) in human embryonic kidney cells transfected with full length human ABCG2, while no transport by cells transfected with human ABCB1 was noted. FTC-inhibited transport of phytoporphyrin was also demonstrated in ABCG2-expressing LLC-PK1 pig kidney cells, consistent with the idea that the pig orthologue, like human ABCG2, transports the photosensitizer. ABCG2 expression was confirmed by immunohistochemistry in the hepatocytes of cow, pig and sheep livers. We conclude that phytoporphyrin is a substrate for ABCG2 and that the transporter is likely responsible for its biliary excretion.     

Increased toxicity of P-glycoprotein-substrate chemotherapeutic agents in a dog with the MDR1 deletion mutation associated with ivermectin sensitivity

Lymphoma was diagnosed in a 4-year-old spayed female Collie, and treatment with a combination chemotherapy protocol incorporating prednisone, L-asparaginase, vincristine, vinblastine, doxorubicin, and cyclophosphamide was initiated. The dog had signs of gastrointestinal tract toxicosis and myelosuppression after treatment with P-glycoprotein-substrate drugs (vincristine, vinblastine, and doxorubicin) even when dosages were reduced, but did not have signs of toxicosis after treatment with cyclophosphamide, a non-P-glycoprotein-substrate drug, even when administered at the full dosage. It was postulated that a deletion mutation in the canine MDR1 gene (deltaMDR1 295-298) could be responsible for the drug toxicoses in this dog. This mutation has been identified as the cause of a functional P-glycoprotein defect in Collies susceptible to the toxic effects of ivermectin, another P-glycoprotein-substrate drug. The MDR1 genotype of this dog consisted of 1 normal and 1 mutant MDR1 allele. Because P-glycoprotein contributes to renal, biliary, and intestinal excretion of P-glycoprotein-substrate drugs, it is possible that drug excretion was delayed in this patient, resulting in clinical signs of toxicosis.     

A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic agonists

The causal factors of equine anhidrosis have not yet been elucidated but defective electrolyte transport mechanisms in the gland are likely to be involved. To investigate this possibility, experiments were performed on cultured equine sweat gland epithelia from five free-sweating UK horses (3 intact males, 2 mares, aged 2-4 years) and from three free-sweating Singapore horses (1 intact male, 2 mares, aged 3-5 years) and three anhidrotic (Singapore) horses (1 intact male, 1 gelding, 1 mare, aged 3-6 years). Cultured cells from each animal were grown on permeable supports and loaded into Ussing chambers to quantify transepithelial resistance and agonist-induced electrolyte transport by the short circuit current (Isc) technique. Transepithelial resistances across the layers of cultured cells were not significantly different between cells from UK and Singapore free-sweating horses, but were significantly reduced in anhidrotic animals. Purinergic agonists added to the apical and basolateral aspects of the cultured cells caused similar increases in Isc between the two populations of unaffected cells, but Isc increases were significantly reduced in anhidrotic animals. Beta-adrenergic agonist stimulation of the anhidrotic cell layers failed to elicit any change in Isc. These pilot results not only confirm earlier conclusions from anatomical findings that failure in the secretory process occurs in anhidrosis but also indicate that both of the known ion transport mechanisms are involved. The trigger for these failures warrants further investigation.     

Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines

Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.     

Aquaporin 9 is expressed in the epididymis of immature and mature pigs

Aquaporins (AQPs) are channel proteins that facilitate the transepithelial and bidirectional movement of water. AQP9 is an aquaporin that is expressed in the mammalian epididymis. This water transport contributes to epididymal sperm concentration. This study aimed to examine the morphology of epididymal epithelium in piglets and boars, as well as the expression and immunolocalization of AQP9. The piglets presented an epididymal epithelium in differentiation with principal, basal and apical cells. The cellular population of the epididymal epithelium in boars consisted of principal, basal, apical, clear and narrow cells. The migratory cells known as halo cells were observed in the epididymis of both piglets and boars. AQP9 expression presented differences between piglets and boars. Moderate intensity of AQP9 immunoreaction was observed in the apical border of the epididymal epithelium of the caput and cauda regions in the piglet epididymis. A moderate‐to‐intense reaction for AQP9 was observed in the nuclei of epithelial cells of the three epididymal regions in the boar epididymis. The region of the cauda epididymis showed reactivity for AQP9 also in the apical border of the epithelium. It is believed that the AQP9 is already functional in piglets at only 1 week of age and is more active, playing a pivotal role in the caput and cauda regions of the epididymis. Moreover, the intense AQP9 expression in the apical border of epithelial cells in the cauda region of the boar epididymis suggests a higher performance of AQP9 in this region, where sperm complete their maturation process, stored and concentrated.     

三聚氰胺和三聚氰酸对蛋鸡毒性效应的研究

本试验旨在探讨饲粮中单独添加三聚氰胺(melanmine,MEL)或与三聚氰酸(cyanuric acid,CYA)合用对蛋鸡健康的影响.将126只78周龄罗曼粉壳蛋鸡随机分为7个组,每组9个重复,每个重复2只鸡.组1为对照组,饲喂基础饲粮;组2～4分别饲喂在基础饲粮中添加MEL 5、15和30 mg/kg的饲粮,组5...     

Expression of P-glycoprotein and breast cancer resistance protein in three cases of canine lymphoma showing drug resistance

In canine lymphoma, drug resistance is the major factor hindering treatment. In this study, we performed immunohistochemical examination of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which are considered as transporters related to multidrug resistance in three recurrent canine lymphomas. All cases were negative for both transporters before anticancer drug administration, but became positive after this administration. The expression was confirmed in capillary endothelial cells, such as in brain capillaries acting as the blood-brain barrier (BBB). It is suggested that both transporters expressed on capillary endothelial cells in lymphoma tissue may inhibit the spread of anticancer drugs into tumor tissues from blood, the same as the BBB. Therefore, capillary endothelial cells could act as a blood-tumor barrier, which might be involved in drug resistance in canine lymphoma.     

Caco-2细胞模型及其在营养素小肠吸收机理研究中的应用

Caco-2细胞源自人结肠癌细胞,体外培养时能自发地进行类似肠道细胞的形态学和生化学上的分化,获得许多小肠吸收细胞的特性,如形成微绒毛结构;在细胞表面形成良好的刷状缘;在细胞间形成紧密连接;分泌水解酶以及合成转运糖、氨基酸和药物等的载体转运系统。由培养在微孔滤膜上的Caco-2细胞构建的模型为研究营养素在小肠的吸收机理提供了一个有效且易于操作的实验手段。本文主要综述了Caco-2细胞模型的建立、特征、检测及其在氨基酸、维生素、核苷和微量元素等营养素小肠吸收机理研究中的应用。     

Polymorphisms and the antiviral property of porcine Mx1 protein

We determined the cDNA sequences of the type I interferon-inducible proteins, pig Mx1 from PK(15) and LLC-PK1 cells, and compared the antiviral activities of both Mx proteins, including Mx1 polymorphisms against vesicular stomatitis virus (VSV). Mx1 cDNA derived from PK(15) cells had an 11 bp-deletion in the 3' end of the coding region, and was estimated to encode 8 amino acid substitutions and a 23 amino acid extension compared to that from LLC-PK1 cells. VSV replication was inhibited in the 3T3 cells expressing Mx1 mRNA after the cDNA was transfected. However, the efficiency of this inhibition was not different between the cells expressing Mx1 mRNA from both PK and LLC. These results indicate that pig Mx1 protein confers resistance to VSV.     

Absorption of horseradish peroxidase by neonatal pig intestinal epithelium: effect of Escherichia coli (055B5) on absorption.

The pathway of macromolecular transport through the neonatal pig small intestinal epithelium was examined, utilizing the cytochemical marker horseradish peroxidase (HRP). The marker was found adsorbed to the apical microvillous of the enterocytes, within apical tubules, and cytoplasmic vacuoles. Extracellular absorbed marker assumed a spherical appearance within the subepithelial spaces but was dispersed within the capillary lumens. Uptake of HRP into the enterocyte occurred in 48-hour old neonatal pigs, but transport into the circulation was not observed. Adherence of Escherichia coli to the surface of the ileal enterocyte did not totally inhibit HRP uptake. The E coli adhered to the surface of the enterocyte or within intercellular vacuoles appeared to be static in as much as they were not involved in the transepithelial migration of envacuolated HRP.     

ABCG2 transporter: therapeutic and physiologic implications in veterinary species

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. While P-glycoprotein, the product of the MDR1 (ABCB1) gene, is the most well-characterized ABC transporter, the pharmacological importance of a related transporter, ABCG2, is starting to be realized in veterinary medicine. Based primarily on human and rodent studies, a number of clinically relevant, structurally and functionally unrelated drugs are substrates for ABCG2. ABCG2 is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain and retinal capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, ABCG2 limits oral absorption of substrate drugs and restricts their distribution to privileged sites such as the brain and retina. ABCG2 is also expressed by tumor cells where it functions to limit the intracellular accumulation of cytotoxic agents, contributing to multidrug resistance. Several ABCG2 polymorphisms have been described in human patients, some of which result in altered drug disposition, increasing susceptibility to adverse drug reactions. Additionally, ABCG2 polymorphisms in humans have been associated with disease states such as gout. Feline ABCG2 has recently been demonstrated to have several amino acid differences at conserved sites compared with 10 other mammalian species. These amino acid differences adversely affect transport function of feline ABCG2 relative to that of human ABCG2. Furthermore, these differences appear to be responsible for fluoroquinolone-induced retinal toxicity in cats and may play a role in acetaminophen toxicity as well. Studies in rodents and sheep have determined that ABCG2 expressed in mammary tissue is responsible for the secretion of many compounds (both therapeutic and toxic) into milk. Finally, data in rodent models suggest that ABCG2 may play an important role in regulating a number of physiologic pathways involved in protecting erythrocytes from oxidative damage.     

Molecular and functional identification of organic anion transporter isoforms in cultured bovine mammary epithelial cells (BME-UV)

Mammary epithelial cells express a diversity of membrane transporters including members of organic cation and organic anion (OAT) transporter subfamilies. Four mammal OAT isoforms have been identified: OAT-1, OAT-2, OAT-3, and OAT-4. The pharmacological significance of OAT isoforms has been emphasized because of their role in the movement of a wide variety of substrates across epithelial barriers. The present study identified (molecularly and functionally) bovine OAT isoforms in bovine mammary epithelial (BME-UV) cells. mRNA expression levels of all tested transporters in BME-UV cells were less than expression levels of the corresponding transporters in bovine kidney. Directionality in the flux of P-aminohippuric acid and acetylsalicylate, compounds known to interact with OAT-1 and OAT-2, respectively, across BME-UV monolayers was not observed at the concentrations used in this study. Directionality was, however, observed in the flux of estrone sulfate (EsS). Adding probenecid, penicillin G or nonradiolabeled EsS to the apical donor compartment significantly increased the apical-to-basolateral flux of EsS across the BME-UV monolayer. These results suggest that BME-UV cells express an organic anion transport system, making it a potentially useful model to study the role of this transport system in the mammary epithelial barrier.     

猪丁型冠状病毒在悬浮培养猪肾细胞LLC-PK1上的增殖特性分析

旨在分析猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)在悬浮培养的猪肾细胞LLC-PK1上的增殖特性,为PDCoV灭活疫苗的规模化生产提供细胞材料。采用逐步降血清法优化LLC-PK1细胞悬浮培养工艺;利用有限稀释法筛选PDCoV适应性细胞株;利用间接免疫荧光法鉴定PDCoV对LLC-PK1细胞的感染性;分别对PDCoV接种LLC-PK1悬浮细胞的初始密度、MOI、收毒时间、TPCK胰酶浓度等参数进行优化,确定最佳悬浮培养条件。成功筛选出可高效增殖PDCoV的单克隆悬浮细胞株LLC-PK1Sa,且利用其增殖的PDCoV可特异性的感染LLC-PK1细胞;PDCoV按MOI为10^-3接种于密度为2×10⁶ cells·mL^-1的LLC-PK1Sa细胞,当TPCK胰酶终浓度达到7.5 μg·mL^-1时,接毒后48 h收获的病毒液滴度最高。本研究首次实现了PDCoV在LLC-PK1Sa悬浮细胞中的高效增殖,并对悬浮培养条件进行了初步优化,可为PDCoV灭活疫苗的规模化生产提供理论参考。     

三聚氰酸水平及与三聚氰胺合用对蛋鸡肝肾损伤的研究

研究旨在探讨三聚氰酸(CYA)水平及与三聚氰胺(MEL)合用对蛋鸡肝肾的损伤。采用2×4二因子随机试验设计,MEL设0或30 mg/kg 2个添加水平;CYA设0、10、20、30 mg/kg 4个添加水平。选用产蛋率相近的78周龄罗曼粉壳蛋鸡144只,随机分成8个处理,每个处理9个重复。试验期35 d。结果表明:添加10~30 mg/kgCYA对1~21 d饲料利用率(FCR)有降低趋势(P=0.070);添加MEL显著降低了1~21 d产蛋率(HDLR)(P<0.05)。10~30 mg/kg CYA显著提高了21 d血清尿酸(UA)含量和35 d血清谷丙转氨酶(ALT)活性(P<0.05);添加MEL显著提高了35 d血清UA含量和降低了21 d血清肌酐(CRE)含量(P<0.05)。10~30 mg/kg CYA显著提高了35 d肾脏组织丙二醛(MDA)含量(P<0.05);添加MEL显著提高了肾脏组织21 d MDA,降低了35 d超氧化物歧化酶(SOD)活性(P<0.05)。单独添加10~30 mg/kg CYA蛋鸡肝细胞肿大、出现空泡变性,肾小管上皮细胞肿胀、肾小球及毛细血管充血;10~30 mg/kg CYA与30 mg/kg MEL合用肝肾的损伤加剧。结果表明,30 mg/kg CYA与30 mg/kg MEL合用对蛋鸡肝肾损伤最严重。     

Roles of oxygen radical production and lipid peroxidation in the cytotoxicity of cephaloridine on cultured renal epithelial cells (LLC-PK1)

To clarify the mechanism of cephalosporin nephrotoxicity, the cytotoxic effects of cephaloridine (CER), a nephrotoxic cephalosporin antibiotic, on the pig kidney proximal tubular epithelial cell line (LLC-PK1) were studied in culture. CER increased the content of hydrogen peroxide and decreased the activity of catalase in the treated cells, followed by an increase in the content of lipid peroxide and decreases in both glutathione peroxidase activity and in the non-protein sulfhydryl content. The levels of NADPH-dependent hydrogen peroxide and superoxide anion production by microsomes prepared from LLC-PK1 cells, and by NADPH-cytochrome P-450 reductase purified from the rat renal cortex were significantly increased by paraquat. The production of these molecules was antagonized by p-chloromer-curibenzoate, an inhibitor of NADPH-cytochrome P-450 reductase. On the other hand, CER did not significantly affect the production of hydrogen peroxide or superoxide anions. These results suggested that the cytotoxic effect of CER on cultured LLC-PK1 cells was due to the increases in hydrogen peroxide and lipid peroxide levels and not microsomal oxygen radical production, and that the mechanism of this cytotoxicity is very different from that of paraquat which induces microsomal oxygen radical production.     

Trichomonas gallinae, in comparison to Tetratrichomonas gallinarum, induces distinctive cytopathogenic effects in tissue cultures

In the present study the interaction of three genetically different clonal cultures of Trichomonas gallinae and Tetratrichomonas gallinarum with a permanent chicken liver (LMH) and a permanent quail fibroblast (QT35) cell culture was studied. Proliferation of T. gallinae cells was associated with a disintegration of the cell monolayer. The initial lesions on the LMH monolayer consisted of a progressive accumulation of the flagellate, forming clumps attached to the monolayer. A prolonged incubation time was characterized by appearance of holes in the cell monolayer with accumulation of trichomonads at their periphery. According to the severeness of the monolayer disruption differences among three tested T. gallinae clones were noticed. Furthermore, filtrates obtained either from axenic cultures of T. gallinae or from infected cell cultures produced a cytopathogenic effect similar to the protozoal cells, on both types of cell cultures. However, the destructive effect of the flagellates and their cell-free filtrates was much more pronounced on the LMH monolayer in comparison with the QT35 cells. Furthermore, freshly seeded LMH and QT35 cells suspended in cell-free filtrates of T. gallinae were unable to form a confluent monolayer. In comparison to T. gallinae, clonal cultures of T. gallinarum or their cell-free filtrates produced no effect on both types of monolayers. Interestingly, the cell-free filtrates obtained from both trichomonad species had an effect on the viability of both cell cultures. However, the cytotoxic effect of T. gallinarum filtrates was less severe than that recorded by T. gallinae. Consequently, for the first time a destruction of specified monolayers induced by T. gallinae-free filtrates could be demonstrated.     

In vivo and ex vivo assessment of the interaction between ivermectin and danofloxacin in sheep

The impact of an efflux pump-related interaction between ivermectin and danofloxacin on their intestinal transport (ex vivo) and disposition kinetics (in vivo) was assessed. Eighteen male Corriedale sheep were randomly assigned to one of three groups. Animals in Group A received 0.2mg/kg ivermectin by SC injection, those in Group B were given 6 mg/kg danofloxacin SC on two occasions 48 h apart and those in Group C were treated with both compounds at the same rates. Plasma concentrations of ivermectin and danofloxacin were measured by HPLC using fluorescence detection. Ex vivo intestinal drug transport activity was measured by the use of the Ussing chamber technique. Plasma concentrations of ivermectin in the first 6 days after injection tended to be higher in Group C than Group A. Contemporaneous treatment with ivermectin significantly increased systemic exposure to danofloxacin (AUC values were 32-35% higher) and prolonged the elimination half-life of danofloxacin (40-52% longer). Ex vivo, incubation with ivermectin significantly decreased the efflux transport of rhodamine 123, a P-glycoprotein substrate, in sheep intestine, but no significant effect of danofloxacin on transport activity was observed. Evaluation of the interaction of danofloxacin with the breast cancer resistance protein (BCRP) showed that pantoprazole and ivermectin significantly decreased danofloxacin secretion in the rat intestine. Thus, the ivermectin-induced reduction of danofloxacin efflux transport observed in this study may involve BCRP activity but the involvement of P-glycoprotein cannot be ruled out.