The cytochrome P450 system of Atlantic salmon (Salmo salar): II. Variations in hepatic catalytic activities and isozyme patterns during an annual reproductive cycle

A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5–7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17 was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b₅, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b₅, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17 levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17 as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17 independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goks\/oyr 1989).     

Role of molecular species catabolism in the temperature-induced restructuring of phosphatidylcholines in liver microsomes of thermally-acclimated rainbow trout (Oncorhynchus mykiss)

Most previous studies of the temperature-induced restructuring of phospholipid molecular species composition have examined steps in the biosynthesis of phospholipids to explain the accumulation of unsaturated fatty acids in membranes of cold-acclimated poikilotherms. In contrast, the present study explores the role of phospholipases in this restructuring process by determining the rates of degradation of specific molecular species of phosphatidylcholine, using enzymes (microsomes) freshly isolated from the liver of rainbow trout. (Oncorhynchus mykiss) acclimated to either 5° or 20°C. The substrate preparation employed to assay phospholipase activity possessed a range of molecular species, all radiolabeled with 1-¹⁴C-palmitic acid at thesn-1 position, similar to that present in native trout liver microsomes. After defined periods of incubation (120 and 240 min at 5°C; 60 and 120 min at 20°C), phospholipids were extracted from the reaction mixture and the distribution of radioactivity among the molecular species of phosphatidylcholine was determined by HPLC/liquid scintillation counting. In general, molecular species catabolism was not significantly influenced by either assay or acclimation temperature. Only in 20°C-acclimated fish did a reduction in assay temperature (from to 20 to 5°C) result in significantly increased proportions of radioactivity being recovered in one polyunsaturated fatty acid-containing species (16:0/22:6-PC). It is concluded: 1) that phospholipase specificity, assayed under conditions approximating thosein situ, is not significantly influenced by temperature; and 2), that the increased proportions of unsaturated fatty acid-containing molecular species of phosphatidylcholine observed at low temperatures must reflect the specificity of biosynthetic rather than degradative processes.     

In-vitro Metabolism of a Novel Monocrotophos Derivative by Rat and Japanese Quail

NADPH-dependent inhibition of hepatic microsomal carboxylesterase by a derivative of monocrotophos (coded as RPR-5) was studied in rat and Japanese quail as a measure of monooxygenase-catalysed activation of RPR-5. There was NADPH-dependent inhibition of hepatic microsomal α-naphthyl acetate esterase (carboxylesterase) both in rat and quail, indicating monooxygenase-catalysed formation of an oxon that subsequently phosphorylated α-NaE. The pattern of in-vitro metabolism of ¹⁴C-labelled RPR-5 by 11000g supernatant (11-S), microsomes and 105000g supernatant (105-S) fractions of rat and quail livers suggested the involvement of microsomal monooxygenases and carboxylesterases. A radiolabelled metabolite (M2) was tentatively identified as an acid produced by carboxyl esterase attack. In rat, metabolism by microsomal and cytosolic (105-S) carboxylesterases appeared to predominate with relatively little oxidative metabolism. In quail, putative microsomal carboxylesterase hydrolysis of RPR-5 was much lower than in the rat with almost neglible hydrolysis by cytosolic fractions. Also, production of M₂ by quail microsomes was substantially reduced after addition of NADPH, suggesting inhibition of a carboxyl esterase by the oxon of RPR-5. Differences in this detoxification of RPR-5 between rat and quail may be an important factor in determining selective toxicity and the results underline the importance of relating metabolism to toxicity when selecting animal models for toxicity testing.     

高效液相色谱法测定大鼠肝微粒体温孵体系中血根碱的含量

本研究建立了大鼠肝微粒体温孵体系中血根碱的高效液相色谱含量测定方法。采用Shimadzu VP-ODS (150 mm×4.6 mm,5 μm)为色谱柱,以乙腈-0.1%磷酸水溶液(30∶70,V/V)为流动相,以1 mL/min流速进样量10 μL在284 nm波长下进行检测,结果显示500和1000 nmol/L血根碱对照品溶液的方法回收率分别为98.3％和98.9%;250、500、1000 nmol/L标准溶液的日内RSD分别为3.76%、2.88%和3.56%,日间RSD分别为5.19%、4.96%和3.84%;血根碱的摩尔浓度在250~10000 nmol/L内与峰面积呈良好的线性关系,表明该方法简单、快速、灵敏度高、特异性好,可用于血根碱在肝微粒体温孵体系中的含量测定。     

鸡传染性贫血免疫对肝微粒体抗氧化酶活性的影响

目的是观察鸡传染性贫血多次免疫对肝微粒体中抗氧化酶活性的影响.对20只SPF鸡随机分为2组,每组10只,免疫组鸡用鸡传染性贫血弱毒苗免疫4次,每次间隔2周,对照组鸡注射同剂量的生理盐水.最后一次免疫后10d取肝脏制备微粒体,利用测试盒测定肝微粒体中的GSH-Px活性、SOD活性、CAT活性和MDA含量.结果与对照组相比,免疫组肝微粒体中GSH Px活性、SOD活性和CAT活性都显著提高(P＜0.05),MDA含量显著减低(P＜0.05).结论为鸡传染性贫血多次免疫可提高鸡体抗氧化能力.     

Reaction phenotyping of vinblastine metabolism in dogs

Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre‐incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre‐incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.     

重组CYP3A46细胞微粒体体外药物 代谢动力学分析

通过对巴马小型猪CYP3A46 重组细胞微粒体体外药物代谢动力学特征比较分析,为巴马小型猪应用 于临床前药理试验提供科学依据。分析重组CYP3A4、CYP3A46 细胞微粒体药物代谢动力学参数Vmax、Km(S50）、 Clint 及IC50 均表明：CYP3A46 的硝苯地平、睾酮的代谢活性及酮康唑的抑制活性均比较接近CYP3A4(P>0.05）, CYP3A46 是否是CYP3A4 的同源酶需要进一步研究确定。     

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanism-based inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6.     

In vitro Metabolism of 14C-Moxidectin by Hepatic Microsomes from Various Species

Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. No data were available on its metabolism in wild species or in monogastrics. The in vitro metabolism of ¹⁴C-moxidectin was studied using hepatic microsomes from several different species: cow (Bos taurus), sheep (Ovis ovis), goat (Capra hircus), deer (Cervus dama), rat (Rattus norvegicus), pig (Sus scrofa and rabbit (Oryctolagus cuniculus). After separation and quantification by HPLC, the extent of metabolism of ¹⁴C-moxidectin was greatest with microsomes from sheep (32.7%) as compared to those from cows (20.6%), deer (15.4%), goats (12.7%), rabbits (7.0%) or rats (3.0%). The least metabolism occurred with microsomes from pigs, with 0.8% of total detected metabolites. A C₂₉ monohydroxymethyl metabolite was detected in the greatest amounts, providing 0.4% out of the total detected radioactivity in pigs and 19.3% in sheep. In addition, the importance of P450 3A in the metabolism of ¹⁴C-moxidectin was confirmed by using in vivo induced P450 in combination with various P450 inhibitors.