Benzimidazole drugs and modulation of biotransformation enzymes

Benzimidazole drugs (e.g., anthelmintics albendazole, fenbendazole, oxfenbendazole, thiabendazole, mebendazole; inhibitors of proton pump omeprazole, lansoprasole, pantoprasole) represent substances used in both human and veterinary medicine; however, from the point of view of induction and inhibition of biotransformation enzymes, research has been carried out mainly due to the initiative of human pharmacologists. The purpose of the present review is to inform about inductive and inhibitive effects of benzimidazole drugs in man, animals and cell cultures. Pharmacological and toxicological consequences of modulation of biotransformation enzymes are discussed and the significance of studies in the field of modulation of biotransformation enzymes in food-producing animals is explained. Since the modulating effect of benzimidazoles strongly varies depending on structure of the individual substances, the particular attention is paid to structure-modulation relationships.     

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.     

The effects of benzimidazole anthelmintics on P4501A in rat hepatocytes and HepG2 cells

Benzimidazole anthelmintics including albendazole, fenbendazole, and mebendazole are widely used in veterinary medicine. The effects of these benzimidazoles on cytochrome P4501A were investigated in primary cultures of rat hepatocytes and in the HepG2 cell line. After incubation of rat hepatocytes and HepG2 for 24-, 48-, and 72-h cells with drugs at various concentrations (0.1-50 microM), the enzyme activities associated with P4501A1/2 (7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation) were measured. The P4501A1/2 protein levels in both model systems were determined by Western blotting. Although all benzimidazoles provoked a significant increase of P4501A1/2 protein levels and P4501A activities, large differences in the induction response were found which was dependent on drug structure, concentration, and model system used. Based on the results, relationships between induction potency and structure of drug were demonstrated, as well as differences between the in vitro systems used. Therefore, pharmacological and toxicological consequences of cytochrome P4501A induction by benzimidazole drugs should be taken into account in veterinary therapy.     

Activities of biotransformation enzymes in pheasant (Phasianus colchicus) and their modulation by in vivo administration of mebendazole and flubendazole

Basal activities of certain pheasant hepatic and intestinal biotransformation enzymes and modulation of their activities by anthelmintics flubendazole (FLBZ) and mebendazole (MBZ) were investigated in subcellular fractions that were prepared from liver and small intestine of control and FLBZ or MBZ treated birds. Several oxidation, reduction and conjugation enzyme activities were assessed. In the liver, treatment of pheasants by FLBZ or MBZ caused very slight or no changes in monooxygenase activities and conjugation enzymes. More significative changes were detected in small intestine. Metyrapone and daunorubicin reductase activities were increased by both substances in the liver. This is the first evidence that certain benzimidazoles modulate reductases of carbonyl group. With respect to the relatively slight extent of the changes caused by FLBZ or MBZ we can assume that repeated administration of therapeutic doses of both FLBZ and MBZ has probably no serious influence on pheasant biotransformation enzyme system.     

The effects of albendazole and its metabolites on hepatic cytochromes P450 activities in mouflon and rat

Albendazole (ABZ) is a benzimidazole anthelmintic widely used in veterinary medicine. The effects of ABZ on cytochromes P450 were investigated in primary cultures of mouflon (Ovis musimon) and rat (Rattus norvegicus) hepatocytes. Besides ABZ, its two main metabolites (albendazole-sulphoxide, ABZSO and albendazole-sulphone, ABZSOO) were tested to clarify which compound is responsible for the induction potency of this benzimidazole drug. After 48 h incubation of hepatocytes with benzimidazoles (0.2-25 microM), ethoxyresorufin O-deethylation (EROD) and benzoxyresorufin O-dearylation (BROD) were measured and the P4501A and 3A protein levels were determined by Western blotting. All benzimidazoles provoked a significant increase of EROD and BROD activities in rat hepatocytes. ABZSO and ABZSOO seemed to be responsible for the induction effect of ABZ on P450s in rat. In mouflon, no pharmacologically significant induction of EROD and BROD activities by benzimidazoles tested was observed. From this point of view, anthelmintic therapy of mouflons with ABZ seems to be safe.     

Modulation of electrogenic transport processes in the porcine proximal colon by enteric neurotransmitters

The aim of our study was to evaluate the involvement of essential pro- and antisecretory neurotransmitters in regulation of secretion in porcine proximal colon. Choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM) and neuropeptide Y (NPY) were located immunohistochemically in the epithelium and subepithelial layer. Modulation of epithelial secretion was studied in Ussing chambers. Application of carbachol (CA), sodium nitroprussid (SNP), VIP and SP but not of NPY or SOM resulted in a chloride dependent increase in short circuit current (I(sc) ). I(sc) increase induced by CA, VIP or SNP was not altered by preincubation with tetrodotoxin or indomethacin. In contrast, SP-induced I(sc) increase was diminished by preincubation with tetrodotoxin, indomethacin, L-nitro-arginin-methyl-ester, and atropine but not hexamethonium. Simultaneous application of CA and VIP, or CA and SNP increased the I(sc) stronger as expected. Applying SP/CA led to a smaller increase in I(sc) as calculated. It is concluded that mainly prosecretory neurotransmitters are involved in regulation of colonic secretion. Cross-potentiations of acetylcholine and nitric oxide and acetylcholine and VIP suggest activation of different intracellular cascades. Similar intracellular pathways may be stimulated by acetylcholine and SP, thus preventing an additive effect of the transmitters.     

Decreased activity of brush border membrane-bound digestive enzymes in small intestines from pigs experimentally infected with porcine epidemic diarrhea virus

Brush border membrane-bound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine aminopeptidase N, and alkaline phosphatase were measured in jejunum from pigs experimentally infected with porcine epidemic diarrhea virus (PEDV). Three piglets from the infected and control groups were euthanized by electrocution and subjected to necropsy at 24, 36, 48, 60, and 72 hours post-inoculation (hpi). The infection of PEDV to jejunum resulted in significant decreases in brush border membrane-bound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine aminopeptidase N, and alkaline phosphatase. PEDV replication results in massive destruction of villous enterocytes leading to a marked reduction of intestinal epithelial surface and brush border membrane-bound digestive enzyme activity. Reduced enzymatic activity and villous atrophy in the small intestine is thought to result in a maldigestive and malabsorptive diarrhea.     

Identification and characterization of the enzymes responsible for the metabolism of the non‐steroidal anti‐inflammatory drugs,flunixin meglumine and phenylbutazone,in horses

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug–drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450‐mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma‐hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5‐OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5‐OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug–drug interactions and identification of reasons for varying pharmacokinetics between horses.     

3D reconstruction of the porcine and equine pulmonary veins,supplemented with the identification of telocytes in the horse

The myocardial sleeve of the porcine and equine pulmonary veins were histologically investigated and reconstructed three dimensionally. Moreover, the localization of neuron cell bodies at the veno‐atrial junction and alongside the myocardial sleeve was light microscopically visualized to depict the organization of nerve, myocardial and fat tissue. Finally, the presence of telocytes inside the equine pulmonary veins was demonstrated by use of transmission electron microscopy. These structures are thought to play a role in the induction of atrial fibrillation, which is frequently seen in horses, while pigs are often used as a cardiovascular model in this context. This data fills in remaining gaps in the literature concerning the histological build‐up of the pulmonary veins wall in pigs and horses. In‐depth knowledge on the myocardial sleeve and its surrounding cell types are important to understand the possible outcome of an ablation therapy as an atrial fibrillation treatment. In pigs and horses, the layout of the pulmonary veins wall concerning these structures is comparable to humans. However, neuron cell bodies were recovered at the veno‐atrial junction in both species but not alongside the myocardial sleeve in horses.     

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100mumol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10mumol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100mumol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.     

Treatment of experimental ureteral strictures by endourological ureterotomy and implantation of stents in the porcine animal model

The objective of this study is to evaluate the dilation of the ureter using endoureterotomy and an expanding-sheath double pigtail ureteral stent in the treatment of experimentally induced ureteral strictures in the porcine animal model. This is a new treatment in the ureteral strictures resolution in Veterinary Urology, although it is not a common affection, it usually appears as a consequence of ureteritis and in the iatrogenic female genital surgery. The experimental study is design in three phases: induction of experimental stricture, diagnosis and treatment of the stricture and follow-up. We have used 10 healthy Large White female pigs. The internal ureteral diameter was measured prior to laparoscopic ligature stricture induction using retrograde ureteropyelography (RUPG). Experimental stricture was diagnosed 4 weeks after intervention, using RUPG and ultrasound, and treated by endoureterotomy and subsequent placement of a double pigtail ureteral stent, which was removed 6 weeks later. The study finished 4 weeks later with measurement of ureteral diameters using RPUG and ultrasound evaluation. Except in one case, all ureters displayed permanent dilation of the strictured area for 10 weeks after treatment (6 weeks with ureteral stent and 4 more weeks without stent). Finally, this technique proved to be effective in cases of short-length and short-living ureteral strictures, and represents a viable alternative to conventional surgery in animals.     

Effects of leptin and adiponectin on the growth of porcine myoblasts are associated with changes in p44/42 MAPK signaling

We hypothesized that both adiponectin and leptin affect the growth of porcine skeletal muscle cells, with fatty acids acting as modifiers in adipokine action and that both adipokines influence the gene expression of their receptors. Therefore, the objective of this study was to investigate the effects of recombinant adiponectin and leptin on cell number (DNA) and DNA synthesis rate with and without oleic acid supplementation, on cell death, and on key intracellular signaling molecules of proliferating porcine myoblasts in vitro. Moreover, the mRNA expression of genes encoding for the leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) as affected by leptin or adiponectin was examined. Recombinant porcine adiponectin (40 μg/mL) and leptin (20 ng/mL) increased DNA synthesis rate, measured as [³H]-thymidine incorporation (P < 0.01), reduced cell viability in terms of lactate dehydrogenase release (P < 0.05), or lowered DNA content after 24 h (P < 0.05). In adiponectin-treated cultures, oleic acid supplementation increased DNA synthesis rate and reduced cell number in a dose-dependent manner (P < 0.05). Both adiponectin (P = 0.07) and leptin (P < 0.05) induced a transient activation of p44/42 mitogen-activated protein kinase (MAPK) after 15 min, followed by decreases after 60 and 180 min (P < 0.05). Adiponectin tended to increase c-fos activation (P = 0.08) and decreased p53 activation at 180 min (P = 0.03). Both adiponectin and leptin down-regulated the abundance of ADIPOR2 mRNA and, transiently, of LEPR mRNA (P < 0.05). In conclusion, adiponectin and leptin may adversely affect the growth of porcine myoblasts, which is related to p44/42 MAPK signaling and associated with changes in ligand receptor gene expression.     

Risk factors associated with porcine cysticercosis in selected districts of Eastern and Southern provinces of Zambia

To determine the risk factors associated with Taenia solium transmission in humans and pigs in the rural areas of Eastern and Southern provinces of Zambia, a questionnaire was administered in 788 households from 155 villages. Pigs were examined from 800 households. Tongue examination and enzyme-linked immunosorbent assay (Ag-ELISA) for the detection of circulating antigens of T. solium cysticerci were used to measure infection in pigs. A snowballing technique was utilised to select households with pigs. Prevalence of households with pigs infected with T. solium on tongue examination by district ranged from 12.7% to 32.1% with Ag-ELISA having a range of 30.0-51.7%. Of the total number of households visited, 18.8% and 37.6% had at least one pig positive for porcine cysticercosis on tongue examination and Ag-ELISA, respectively. Risk factors associated with T. solium infection were lack of pork inspection at slaughter (96.7%), consumption of pork with cysts (20.1%), selling of pork infected with T. solium cysticerci (18.3%), free-range husbandry system (83.2%) and absence of latrines (58.0). Free-range husbandry system (OR=1.68; 95% CI=1.36-2.07) was a significant risk factor for porcine cysticercosis in the surveyed areas. The result that pigs were mostly kept on free-range and semi-intensive husbandry systems may have permitted them to have access to eating human faeces that could be contaminated with tapeworm eggs. This study has shown that T. solium infection poses a high public health risk in the study areas and urban areas as well. We recommend that a human survey be conducted to verify the human exposure to taeniasis and/or cysticercosis in Zambia.     

Vaccination with a genotype 1 modified live vaccine against porcine reproductive and respiratory syndrome virus significantly reduces viremia,viral shedding and transmission of the virus in a quasi-natural experimental model

The present study assessed the efficacy of vaccination against genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV) in terms of reduction of the transmission. Ninety-eight 3-week-old piglets were divided in two groups: V (n = 40) and NV (n = 58) that were housed separately. V animals were vaccinated with a commercial genotype 1 PRRSV vaccine while NV were kept as controls. On day 35 post-vaccination, 14 NV pigs were separated and inoculated intranasally with 2 ml of a heterologous genotype 1 PRRSV isolate (“seeder” pigs, SP). The other V and NV animals were distributed in groups of 5 pigs each. Two days later, one SP was introduced into each pen to expose V and NV to PRRSV. Sentinel pigs were allocated in adjacent pens. Follow-up was of 21 days. All NV (30/30) became viremic after contact with SP while only 53% of V pigs were detected so (21/40, p < 0.05). Vaccination shortened viremia (12.2 ± 4 versus 3.7 ± 3.4 days in NV and V pigs, respectively, p < 0.01). The 50% survival time for becoming infected (Kaplan–Meier) for V was 21 days (CI_95% = 14.1–27.9) compared to 7 days (CI_95% = 5.2–8.7) for NV animals (p < 0.01). These differences were reflected in the R value as well: 2.78 (CI_95% = 2.13–3.43) for NV and 0.53 (CI_95% = 0.19–0.76) for V pigs (p < 0.05). All sentinel pigs (10/10) in pens adjacent to NV + SP pens got infected compared to 1/4 sentinel pigs allocated contiguous to a V + SP pen. These data show that vaccination of piglets significantly decrease parameters related to PRRSV transmission.     

Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus

The first case of porcine reproductive and respiratory syndrome (PRRS) in Denmark was diagnosed in March 1992 by the detection of specific antibodies against PRRS virus in serum samples originating from sows in a herd located on the island of Als. Subsequently, PRRS virus was isolated from a 200-sow farrow-to-finish herd with clinical signs consistent with PRRS. The virus was isolated by inoculation of pleural fluid from a stillborn piglet onto porcine pulmonary alveolar macrophages. The isolate was identified as PRRS virus by staining with a specific antiserum. By electron microscopy, the virus particle was found to be spherical and enveloped, measuring 45–55 nm in diameter and containing a 30–35 nm nucleocapsid. Only minor antigenic differences were found between the Danish and a Dutch isolate. Following intranasal inoculation of 3 pregnant gilts with the Danish isolate transplacental infection was demonstrated by the re-isolation of PRRS virus from approximately 45% of the piglets from the experimentally infected gilts. However, the experimental infection produced no significant reproductive disorders or other clinical signs. At autopsy, histopathological examination revealed slight interstitial pneumonia in a few piglets.     

CYP19A1调控内源性雌激素合成促进牦牛COCs细胞自噬和早期发育能力

旨在探索牦牛卵母细胞成熟过程中细胞色素P450芳香化酶(cytochrome P450arom, CYP19A1)对内源性雌激素(17β-estradiol, E₂)分泌、卵母细胞自噬和后续胚胎发育能力的影响。本研究在牦牛卵丘卵母细胞复合体(cumulus-oocyte complexes, COCs)体外成熟过程中,分别用等体积生理盐水、最佳浓度E₂(10^-7 mol·L^-1)、CYP19A1诱导剂黄曲霉毒素B1(aflatoxin B1, AFB1)、CYP19A1抑制剂双酚A(bisphenol A, BPA)处理,实时荧光定量PCR(real-time quantitative PCR, qRT-PCR)、Western blot和免疫荧光技术检测各组成熟COCs中CYP19A1表达水平,酶联免疫吸附方法(enzyme-linked immunosorbent assay, ELISA)检测诱导和抑制CYP19A1处理组牦牛成熟COCs培养液中E₂水平;分析不同处理组C...     

Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge

Disease exacerbation was observed in pigs challenged with virulent porcine reproductive and respiratory syndrome virus (PRRSV) following immunisation with a recombinant GP5 sub-unit PRRSV vaccine (rGP5) produced in E. coli. Eighteen animals were divided into three experimental groups: group A were immunised twice IM with rGP5, 21 days apart; group B acted as positive controls (challenged but not immunised); and group C were negative controls. Pigs in groups A and B were challenged 21 days after the second immunisation of the group A animals. Following challenge, three pigs given rGP5 exhibited more severe clinical signs than the positive controls, including respiratory distress and progressive weight-loss. Although not statistically significant, the more severe disease exhibited by group A animals may suggest previous immunisation as a contributory factor. The mechanisms of these findings remain unclear and no association could be established between the severity of disease, non-neutralising antibody concentrations and tissue viral loads.     

Experimental reproduction of porcine respiratory disease complex in pigs inoculated porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae and followed by inoculation with porcine circovirus type 2

The aim of this study was to reproduce severe pneumonic lesions, similar to those during naturally-occurring porcine respiratory disease complex, in pigs dually inoculated with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae at 6 weeks of age, followed by inoculation with porcine circovirus type 2 at two weeks after. Time and sequence of infection with three pathogens mirror Asian field conditions. Microscopically, interstitial pneumonia and peribronchiolar lymphoid hyperplasia are considered the most characteristic lung lesions in infected pigs. The results of the present study demonstrate that inoculation of pigs with these three pathogens can lead to severe interstitial pneumonia with peribronchial or peribronchiolar lymphoid hyperplasia and fibrosis.