The suppressive effects of lipopolysaccharide-induced acute phase response on hepatic cytochrome P450-dependent drug metabolism in rabbits

The acute phase response (APR) was induced by five separate intravenous (i.v.) injections of Escherichia coli lipopolysaccharide (LPS, 17 microg/kg each time) in rabbits, with intervals of 1 h. This model was used to study the effects of APR on the activities of hepatic microsomal cytochrome P450 (CYP)-dependent enzyme including drug metabolism. Five female rabbits were included in each of four groups, a control group and three LPS-treated groups (group I, II and III). The rabbits of the control, group I, II and III were killed at 1, 1, 3 and 7 days after saline (control only) or the LPS injection, respectively. The APR was confirmed by increases in rectal body temperature, plasma concentrations of interleukin-6 and C-reactive protein (CRP). Pharmacokinetics of antipyrine before death were examined in every group. Antipyrine was administered (5 mg/kg) at 24 h (control and group I), 3 days (group II) and 7 days (group III) after the first LPS injection. Total body clearance (Cl(tot)) of antipyrine tended to decrease in group I. All the livers were excised for measuring CYP-dependent activities. Total CYP content and several CYP-dependent activities (aminopyrine N-demethylation, aniline 4-hydroxylation and caffeine 3-demethylation) decreased in group I. The maximum velocity (Vmax) values of those enzymes, and the amount of CYP1A1/1A2 and CYP2E1 apoproteins appeared to decrease. Michaelis constant (Km) values of those enzymes were not affected by the APR. Rectal body temperature recovered to normal at 3 days after the first LPS injection in group II and III. The concentration of CRP, albumin, total CYP content and the plasma clearance of antipyrine returned to the control levels at 7 days after the first LPS injection. These results suggest that the metabolism of drugs, including CYP-dependent drug metabolizing activity, is suppressed markedly in incipient APR induction in rabbits, and the drug metabolizing capacity is returned to normal at 7 days after APR induction.     

Feed–drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens

The expression of hepatic drug‐metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition‐derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed–drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP‐inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non‐specific CYP‐inducer, simultaneously with two different doses of intra‐ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB‐triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP‐inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP‐inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition.     

In vitro complex formation and inhibition of hepatic cytochrome P450 activity by different macrolides and tiamulin in goats and cattle

In humans, clinically relevant drug-drug interactions occur with some macrolide antibiotics via the formation of stable metabolic intermediate (MI) complexes with enzymes of the cytochrome P4503A (CYP3A) subfamily. The formation of such complexes can result in a decreased biotransformation rate of simultaneously administered drugs. In previous studies it was shown that the veterinary antibiotic tiamulin was also able to form a stable MI complex in pigs and rats. In the present study the relative CYP3A inhibiting potency and MI complex formation of a series of macrolide antibiotics and tiamulin were studied in microsomal fractions of goat and cattle and in a cell-line expressing bovine CYP3A. Tiamulin and triacetyloleandomycin (TAO) were found to be effective inhibitors of CYP450 activity in all systems tested. Erythromycin and tilmicosin were found to be relatively less effective inhibitors of CYP450 activity in microsomes, and their activity in the bovine CYP3A4 expressing cell line was relatively weak. Tylosin was shown to be a weak inhibitor in microsomes and not in the cell line, whereas spiramycin had no effect at all. MI-complex formation measured by spectral analysis was seen with TAO, tiamulin, erythromycin and tylosin, but not with tilmicosin and spiramycin. Although additional factors play a role in vivo, these results may explain potential drug-drug interactions and differences between related compounds in this respect.     

Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse

Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).     

Inhibition of cytochrome P450 activity enhances the systemic availability of triclabendazole metabolites in sheep

Understanding the disposition kinetics and the pattern of metabolism is critical to optimise the flukicidal activity of triclabendazole (TCBZ) in ruminants. TCBZ is metabolised by both flavin-monooxygenase (FMO) and cytochrome P450 (P450) in the liver. Interference with these metabolic pathways may be useful to increase the systemic availabilities of TCBZ metabolites, which may improve the efficacy against Fasciola hepatica . The plasma disposition of TCBZ metabolites was evaluated following TCBZ co-administration with FMO [methimazole (MTZ)] and P450 [piperonyl butoxyde (PB) and ketoconazole (KTZ)] inhibitors in sheep. Twenty (20) healthy Corriedale x Merino weaned female lambs were randomly allocated into four experimental groups. Animals of each group were treated as follow: Group A, TCBZ alone (5 mg/kg, IV route); Group B, TCBZ (5 mg/kg, IV) + MTZ (3 mg/kg, IV); Group C, TCBZ (5 mg/kg, IV) + PB (30 mg/kg, IV) and Group D, TCBZ (5 mg/kg, IV) + KTZ (10 mg/kg, orally). Blood samples were taken over 240 h post-treatment and analysed by HPLC. TCBZ sulphoxide and sulphone were the main metabolites recovered in plasma. MTZ did not affect TCBZ disposition kinetics. TCBZ sulphoxide Cmax values were significantly increased ( P  < 0.05) after the TCBZ + PB (62%) and TCBZ + KTZ (37%) treatments compared to those measured in the TCBZ alone treatment. TCBZ sulphoxide plasma AUCs were higher ( P  < 0.05) in the presence of both PB (99%) and KTZ (41%). Inhibition of TCBZ P450-mediated oxidation in the liver accounted for the increased systemic availability of its active metabolite TCBZ sulphoxide. This work contributes to the search of different strategies to improve the use of this flukicidal drug in ruminants.     

乐果亚急性暴露对大鼠胆碱酯酶浓度及细胞色素P450亚型表达量的影响

对6周龄雄性Wistar大鼠5次灌胃给予20％LD50的乐果，观察给药后大鼠的毒性反应，每日测定实验鼠采食量、饮水量和体重。最后一次给药后第24h，采用丁酰硫代胆碱法测定大鼠红细胞、血清和脑组织中的胆碱酯酶浓度，并采用定量RT-PCR法检测大鼠肝中细胞色素P450基因8个亚型的表达量。结果表明，大鼠用药后不同时间内出现了有机磷药物中毒的典型症状，其采食量、饮水量和体重明显下降，红细胞、血清和脑组织中胆碱酯酶浓度均明显低于对照组，肝细胞色素P450的8个亚型中CYP2B1／2的表达量明显高于对照组。     

A lipopolysaccharide-induced acute phase response in the pig is associated with a decrease in hepatic cytochrome P450-mediated drug metabolism

Drug disposition, including hepatic drug metabolism, is markedly affected by infection, inflammation and other conditions that invoke the acute phase response. In the present study, an Escherichia coli lipopolysaccharide (LPS)-induced acute phase response model was developed in pigs. This model was used to study the effects of the acute phase response on drug disposition and hepatic drug metabolism in vivo and in microsomal preparations. The results obtained were compared with those from Actinobacillus pleuropneumoniae -infected pigs. Intermittent intravenous administration of LPS induced a mild acute phase response as evidenced by increased rectal body temperatures, anorexia and increased cytokine (TNF-α and IL-6) serum levels within 1-2 h after the first LPS injection. The acute phase response is associated with a pronounced decrease of antipyrine plasma clearance (control 8.5 ± 0.8 vs. LPS 2.2 ± 0.7 mL/min.kg). Furthermore, total cytochrome P450 content and microsomal cytochrome P450-dependent activities were significantly decreased after 24 h. The decrease in cytochrome P450 activities was accompanied by losses of cytochrome P4501A and P4503A apoproteins. The microsomal glucuronidation rate of 1-naphthol was not affected in LPS-treated pigs. Comparing the LPS model with our previous findings in the Actinobacillus pleuropneumoniae model showed a remarkable similarity with regard to the effects on hepatic drug metabolism.     

Effect of oral administration of low doses of pentobarbital on the induction of cytochrome P450 isoforms and cytochrome P450-mediated reactions in immature Beagles

OBJECTIVE: To determine the effect of oral administration of low doses of pentobarbital on cytochrome P450 (CYP) isoforms and CYP-mediated reactions in immature Beagles. ANIMALS: 42 immature (12-week-old) Beagles. PROCEDURE: Dogs were grouped and treated orally as follows for 8 weeks: low-dose pentobarbital (50 microg/d; 4 males, 4 females), mid-dose pentobarbital (150 microg/d; 4 males, 4 females), high-dose pentobarbital (500 microg/d; 4 males, 4 females), positive-pentobarbital control (10 mg/kg/d; 2 males, 2 females), positive-phenobarbital control (10 mg/kg/d; 2 males, 2 females), and negative control (saline 10.9% NaCl] solution; 5 males, 5 females). Serum biochemical and hematologic values were monitored. On necropsy examination, organ weights were determined, and histologic evaluation of tissue sections of liver, kidney, small intestine, testes, epididymis, and ovaries was performed. Hepatic and intestinal drug-metabolizing enzyme activities were measured, and relative amounts of CYP isoforms were determined by western blot analysis. RESULTS: The amount of a hepatic CYP2A-related isoform in dogs from the high-dose pentobarbital treatment group was twice that of dogs from the negative control group. CYP2C was not detectable in small intestinal mucosa of dogs from the negative control group; measurable amounts of CYP2C were found in dogs from the various (low-, mid-, and high-dose) pentobarbital treatment groups and from positive-pentobarbital and positive phenobarbital control groups. Several CYP-mediated reactions increased in a dose-dependent manner. The lowest calculated effective dose of pentobarbital ranged from 200 to 450 microg/d. CONCLUSIONS AND CLINICAL RELEVANCE: Several CYP isoforms and their associated reactions were induced in dogs by oral administration of low amounts of pentobarbital.     

Comparative study of three intramuscular anaesthetic combinations (medetomidine/ketamine, medetomidine/fentanyl/midazolam and xylazine/ketamine) in rabbits

OBJECTIVE: To compare the quality of surgical anaesthesia and cardiorespiratory effects of three intramuscular (IM) anaesthetic combinations in rabbits. STUDY DESIGN: Prospective randomized cross-over experimental study. ANIMALS: Nineteen adult female chinchilla mixed-bred rabbits weighing 3.9 +/- 0.8 kg. METHODS: Rabbits were given one of three IM anaesthetic combinations: 0.25 mg kg(-1) medetomidine and 35.0 mg kg(-1) ketamine (M-K), 0.20 mg kg(-1) medetomidine and 0.02 mg kg(-1) fentanyl and 1.0 mg kg(-1) midazolam (M-F-Mz) and 4.0 mg kg(-1) xylazine and 50 mg kg(-1) ketamine (X-K). The effects of anaesthesia on nociceptive reflexes, circulatory and respiratory function were recorded. Statistical analyses involved repeated measures anova with paired Student's t-test applied post hoc. P-values <0.05 were considered as significant. RESULTS: Reflex loss was most rapid and complete in M-K recipients, whereas animals receiving M-F-Mz showed the longest tolerance of endotracheal intubation (78.1 +/- 36.5 minutes). Loss of righting reflex was significantly most rapid (p < 0.05) in the X-K group (114.7 +/- 24.0 minutes). Surgical anaesthesia was achieved in 16 of 19 animals receiving M-K, in 14 animals receiving M-F-Mz, and in seven animals with X-K, but only for a short period (7.1 +/- 11.6 minutes). This was significantly (p < 0.001) shorter than with M-K (38.7 +/- 30.0 minutes) and M-F-Mz (31.6 +/- 26.6 minutes). Heart rates were greatest in X-K recipients; lowest HR were seen in animals receiving M-F-Mz. Mean arterial blood pressure was significantly higher (about 88 mmHg) during the first hour in the M-K group. During recovery, the greatest hypotension was encountered in the X-K group; minimum values were 53 +/- 12 mmHg. Six of 19 animals in the M-F-Mz group showed a short period of apnoea (30 seconds) immediately after endotracheal intubation. Respiratory frequency was significantly lower in this group (p < 0.001). Highest values for arterial carbon dioxide partial pressures (PaCO(2)) (6.90 +/- 0.87 kPa; 52.5 +/- 6.5 mmHg) occurred after induction of anaesthesia in group M-F-Mz animals. There was a marked decrease in PaO(2) in all three groups (the minimum value 5.28 +/- 0.65 kPa [39.7 +/- 4.9 mmHg] was observed with M-K immediately after injection). Arterial PO(2) was between 26.0 and 43.0 kPa (196 and 324 mmHg) in all groups during O(2) delivery and decreased - but not <7.98 kPa - on its withdrawal. Immediately after drug injection, pH(a) values fell in all groups, with lowest values after 30 minutes (7.23 +/- 0.03 with M-K, 7.28 +/- 0.05 with M-F-Mz, and 7.36 +/- 0.04 with X-K). The X-K animals showed significantly (p < 0.001) higher pH values than medetomidine recipients. During 1 hour of anaesthesia pH values in the medetomidine groups remained below those of the X-K group. CONCLUSIONS: Surgical anaesthesia was induced in most animals receiving medetomidine-based combinations. Arterial blood pressure was maintained at baseline values for about 1 hour after M-K. Transient apnoea occurred with M-F-Mz and mandates respiratory function monitoring. Oxygen enrichment of inspired gases is necessary with all three combinations. Endotracheal intubation is essential in rabbits receiving M-F-Mz. CLINICAL RELEVANCE: The quality of surgical anaesthesia was greatest with M-K. All combinations allowed recoveries of similar duration. It is theoretically possible to antagonize each component of the M-F-Mz combination.     

Gender-related differences in cytochrome P450 in porcine liver--implication for activity, expression and inhibition by testicular steroids

In pigs, the hepatic cytochrome P450 (CYP) 1A2, 2A and 2E1 activity is important in the regulation of skatole accumulation in adipose tissue. This study investigated gender-related differences in CYP1A2, 2A and 2E1 dependent activity, protein and mRNA expression. This study also investigated the gonadal steroid dependent inhibition of CYP activity in relation to gender and dietary composition. Microsomes were prepared from the liver of female and entire male pigs (Landrace × Yorkshire sire and Duroc boars) reared under similar conditions and slaughtered at an age of 164 days. A group of entire male pigs fed dried chicory root for 16 days prior to slaughter were included in the study. CYP activities were assessed by the use of probe substrates, whilst mRNA and protein expression were analysed by RT-PCR and Western blotting. Furthermore inhibition of CYP dependent activity by gonadal steroids was assessed in vitro. Microsomes from female pigs had greater CYP1A2 and 2A activity, as well as mRNA expression compared to entire male pigs. The antibodies used did not detected differences in protein expression. In vitro inhibition by 17β-oestradiol, oestrone, androstenone and 3β-OH androstenol of CYP2E1 activity in microsomes from entire male pigs as well as inhibition of CYP1A activity in chicory fed entire male pigs was observed. Apart from that no effect of steroids was shown. In conclusion, female pigs show greater CYP activity and mRNA expression. Including chicory in the diet for 16 days changed the gonadal steroid dependent inhibition of CYP activity in entire male pigs.     

cDNA cloning and expressions of cytochrome P450 1A in zebrafish embryos

Cytochrome P450 1A (CYP1A) is well known for being induced by aromatic hydrocarbons, including 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). We determined the complete cDNA sequence of a CYP1A open reading frame with both 5'- and 3'-ends in zebrafish (zfCYP1A), a useful model for environmental toxicology. zfCYP1A shows high percentage identity with CYP1As of mammals, domestic fowl and xenopus (51.9-60.4%), as well as the other fish species (63.8-89.2%). As revealed by in situ hybridization and immunohistochemistry, zfCYP1A was scarcely detected in control embryos but was markedly induced by TCDD especially in heart, vascular endothelial cells, intestinal epithelium, pronephros and outer integument in both prehatched and hatched embryos. These expression patterns are consistent with possible involvement of zfCYP1A in TCDD-induced toxicities.     

氟苯尼考对鲫鱼CYP2E1活性影响的药代动力学评价

采用以氯唑沙宗为底物探针的药代动力学方法研究氟苯尼考对鲫鱼细胞色素P450 2El活性的影响。试验组鲫鱼每日经口给予氟苯尼考100mg／kg（按体重给药，下同），连续5d后，与对照组鲫鱼同时一次性腹腔注射氯唑沙宗10mg／kg，用高效液相色谱法测定氯唑沙宗在鲫鱼体内不同时间点的血药浓度，并计算药代动力学参数。结果表明，氯唑沙宗在两组鲫鱼体内代谢的药时数据均符合二室模型；与对照组相比，试验组氯唑沙宗的半衰期增加了5倍（P〈0．01），药时曲线下面积增加了3．3倍（P〈0．01），总清除率减少了83．8％（P〈0．01）。提示，氟苯尼考对鲫鱼CYP2E1具有显著的抑制作用。其他药物与氟苯尼考合用时，其有效性和安全性可能会受到影响。     

Effect of oral administration of clinically relevant doses of dexamethasone on regulation of cytochrome P450 subfamilies in hepatic microsomes from dogs and rats

OBJECTIVE: To evaluate the effect of oral administration of dexamethasone (DEX) at clinically relevant doses on metabolic activities of cytochrome P450 (CYP) isoenzymes in dogs and rats. ANIMALS: 15 healthy 1-year-old male Beagles and 20 healthy 10-week-old male Wistar rats. PROCEDURE: Hepatic microsomes were harvested from dogs treated orally with DEX at 2.5 and 7.5 mg for 5 days and from rats treated orally with DEX at 0.75, 6, and 48 mg/kg for 5 days. 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam were used as CYP1A, CYP2C, CYP2D, and CYP3A substrates, respectively. Concentrations of metabolites formed by CYPs were measured by use of high-performance liquid chromatography, except for the resorufin concentrations measured by use of a fluorometric method. Reaction velocity-substrate concentration data were analyzed to obtain maximum reaction velocity (Vmax) and Michaelis-Menten constant (Km). RESULTS: Values of Vmax for midazolam 4-hydroxylation were significantly decreased by treatment with DEX at 2.5 and 7.5 mg in dogs, although values of Km were not affected. Values of Vmax for bufuralol 1'-hydroxylation were also decreased by treatment with DEX. In rats, values of Vmax for midazolam 4- hydroxylation were significantly decreased by treatment with DEX at 0.75 and 6 mg/kg but significantly increased at 48 mg/kg. Other reactions were not affected by treatment with DEX. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that DEX downregulates the CYP3A subfamily when administered at clinically relevant doses to dogs. The effect of downregulation of CYP3A in dogs treated with DEX should be considered to avoid adverse effects from coadministration of drugs.     

几种临床常用药物对猪肝细胞色素P450酶系的影响

研究了几种临床常用药物对猪肝微粒体细胞色素P450酶系的影响。红霉素、环丙沙星、地塞米松和乙醇,按5倍治疗剂量进行肌肉注射每天1次,连续给药10 d。差速离心法制备猪肝微粒体,B radford法测定蛋白浓度,分光光度法检测6种肝细胞色素P450酶含量及活性。结果表明,4种药物除环丙沙星外,均对P450酶的总活性产生显著影响(P<0.05),同时地塞米松组的微粒体蛋白含量和红霉素-N-脱甲基酶活性,乙醇组的苯胺-4-羟化酶活性和红霉素组的红霉素-N-脱甲基酶活性,与对照组相比均差异显著(P<0.05)。说明环丙沙星和红霉素对猪肝细胞色素P450酶系有选择性抑制作用,而地塞米松和乙醇对其起选择性诱导作用。