离体鼠肝细胞及P—450酶系与拟除虫菊酯类杀虫剂的的相互作用

经研究表明顺式氯氰菊酯．甲氰菊酯、氟氰菊酯、氰戊菊酯在正辛醇－水相中的分配系数分别为６．１３，５．５０，５．５５和４．０２。离体培养的大白鼠原代肝细胞上述杀虫（氟氰菊酯外）的吸收系数（ｑ值）分别为３２２．６，２８１．１，２０１．９。上述杀虫剂在离体培养的原代肝细胞中降解很快，湿育３０分钟后降解速率分别为９０．０％（顺式氯氰菊酯）、８６．２％（甲氰菊酯）、６９．５％（氰戊菊酯）。同时有明显的减少离体     

拟除虫菊酯的结构与害虫抗药性的关系

以家蝇Musca domestica vicina L.为试虫,用汰选方法研究了家蝇对六种不同拟除虫菊酯的抗性发展.结果表明,家蝇对不同化学结构和不同光学异构体组分的拟除虫菊酯抗性差别很大.对溴氧菊酯、顺式氯氰菊酯抗性发展很快,氯氰菊酯次之,氰戊菊酯、氰戍菊酯A和氰戊菊酯A_σ抗性发展较慢.用抗溴氰菊酯家蝇品系和点滴法测定了十种拟除虫菊酯和七种有机磷杀虫剂的毒力,讨论了它们之间的交互抗性和结构与抗性的关系.溴氰菊酯(抗性比值24.00)、氯氰嫡酯(抗性比值20.11)、顺式氯氰菊酯(抗性比值38.10)和二氯苯醚菊酯(抗性比值11.04),结构相近的交互抗性比较严重.氰戊菊酯(抗性比值4.64)、氰戊菊酯A(抗性比值5.97)、戊菊酯(抗性比值4.49)和氟氰菊酯(抗性比值4.12)化学结构中醇部分与溴氰菊酯相同、酸部分不同,它们与溴氰菊酯交互抗性水平较低.联苯菊酯(抗性比值1.98)化学结构中酯和醇部分都与溴氰菊酯不同,其交互抗性水平较低,杀螟松等七种有机磷杀虫剂,除敌敌畏与溴氰菊酯有轻微交互坑性外,其它均无交互抗性.     

甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性机理

通过对活体增效作用进行测定和生化分析,探讨了甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性机理.结果表明:增效醚(PBO)、增效磷(SV1)、磷酸三苯酯(TPP)和顺丁烯二酸二乙酯(DEM)对甜菜夜蛾抗氰戊菊酯品系(Fen-R)和敏感品系(S)的增效倍数之比分别为10.2、7.8、12.5和1.1,对抗顺式氯氰菊酯品系(Cyp-R)和敏感品系(S)的增效倍数之比分别为21.6、15.5、8.6和1.2.PBO、SV1和TPP对氰戊菊酯和顺式氯氰菊酯均有显著增效作用,表明多功能氧化酶和羧酸酯酶均参与了甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性.Fen-R品系和Cyp-R品系4龄幼虫羧酸酯酶的活性分别是S品系的1.9和2.2倍,而谷胱甘肽-S-转移酶活性与S品系差异不显著,表明羧酸酯酶活性的提高是甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯产生抗性的重要原因,谷胱甘肽-S-转移酶与两种药剂的抗性无关.Fen-R品系和Cyp-R品系的Na-K-ATPase活性与S品系均无显著差异,但在相同浓度下氰戊菊酯和顺式氯氰菊酯对S品系Na-K-ATPase的抑制作用显著高于抗性品系,表明抗性品系Na-K-ATPase对杀虫剂的敏感性已明显降低.     

甜菜夜蛾对三种拟除虫菊酯类杀虫剂的抗性遗传力及风险评估

在室内抗性选育的基础上,应用数量遗传学中的域性状分析法,分别研究了甜菜夜蛾(Spodoptera exigua)对三氟氯氰菊酯、氰戊菊酯和顺式氯氰菊酯3种拟除虫菊酯类杀虫剂的抗性现实遗传力(h²),并对3种药剂在不同杀死率下的抗性发展速率进行了预测.结果表明,用三氟氯氰菊酯、氰戊菊酯、顺式氯氰菊酯分别连续汰选12、10和10代后,甜菜夜蛾对3种药剂的抗性分别提高了18.1、27.6和45.4倍,抗性现实遗传力分别为0.2567、0.3571和0.4239.假设遗传力为实验室汰选估算值的一半、药剂杀死率为50%～90%、预计抗性增长10倍时,三氟氯氰菊酯需要约9～20代,氰戊菊酯需要约6～14代,顺式氯氰菊酯需要约5～12代.甜菜夜蛾对以上3种拟除虫菊酯类杀虫剂均存在抗性风险.相比之下,顺式氯氰菊酯和氰戊菊酯的抗性风险大于三氟氯氰菊酯.     

我国白纹伊蚊对三种拟除虫菊酯类杀虫剂抗性检测诊断剂量的建立

【目的】建立中国白纹伊蚊Aedes albopictus成蚊对溴氰菊酯、氯菊酯和高效氯氟氰菊酯杀虫剂抗性检测的诊断剂量。【方法】应用溴氰菊酯、氯菊酯和高效氯氟氰菊酯原药制作不同浓度的药膜滤纸,接触筒法测定白纹伊蚊实验室敏感品系成蚊对3种拟除虫菊酯类杀虫剂的敏感性,记录1 h的击倒数和24 h的死亡数。应用Excel 2007和SPSS20.0进行数据统计处理,并制作杀虫剂的毒力回归线,计算各自的LC_(50)和LC_(99)值。以2倍LC_(99)值作为区分抗性和敏感种群的诊断剂量,制作药膜滤纸,接触筒法生物测定海口市白纹伊蚊现场种群成蚊对杀虫剂的抗药性。【结果】溴氰菊酯、高效氯氟氰菊酯和氯菊酯杀虫剂对白纹伊蚊实验室敏感品系成蚊的LC_(50)值分别为0.00619%,0.01403%和0.05009%,LC_(99)值分别为0.05175%,0.11859%,和0.53165%,相对应的诊断剂量分别为0.1035%,0.2372%和1.0633%。应用上述溴氰菊酯、氯菊酯和高效氯氟氰菊酯诊断剂量测定的海口市白纹伊蚊现场种群的死亡率分别为22.58%,36.29%和40.83%,表明该种群对这3种菊酯类杀虫剂均已产生了抗性。【结论】本研究建立的白纹伊蚊对3种拟除虫菊酯类杀虫剂的诊断剂量可作为该蚊成蚊抗药性监测的参考。     

两个广西草地贪夜蛾种群对常用杀虫剂的敏感性测定

为了解入侵广西的草地贪夜蛾Spodoptera frugiperda对不同杀虫剂的敏感性,为筛选该虫的有效防治药剂,以及为该虫的抗药性监测提供科学依据。本研究用浸叶法分别测定了田阳种群对13种杀虫剂和南宁种群对5种杀虫剂的敏感性,并比较了两个种群对5种杀虫剂的敏感性差异。结果表明,田阳种群对13种杀虫剂的敏感性依次为:甲氨基阿维菌素苯甲酸盐多杀菌素氯虫苯甲酰胺虫螨腈溴氰菊酯阿维菌素甲氰菊酯氟氯氰菊酯茚虫威呋虫胺毒死蜱高效氯氰菊酯吡虫啉,其中对甲氨基阿维菌素苯甲酸盐、多杀菌素、氯虫苯甲酰胺的敏感性较高,LC_(50)值分别为0.0015、0.0062、0.0075 mg a.i/L,毒力指数分别为480.47、116.24、96.09,其次是虫螨腈、溴氰菊酯、阿维菌素,LC_(50)值分别为0.0296、0.0590、0.0619 mg a.i/L,毒力指数分别为:24.35、12.22、11.64,对其他药剂的敏感性较低,LC_(50)值在0.2544～0.7207 mg a.i/L,毒力指数在1～2.83,除对溴氰菊酯和阿维菌素、甲氰菊酯和氟氯氰菊酯、呋虫胺和毒死蜱、高效氯氰菊酯和吡虫啉的敏感性差异不显著外,对其它药剂差异显著。南宁种群对5种杀虫剂的敏感性依次为:氯虫苯甲酰胺溴氰菊酯氟氯氰菊酯甲氰菊酯高效氯氰菊酯,LC_(50)值分别为0.0142、0.0413、0.0792、0.2081和0.5947 mg a.i/L,毒力指数分别为:41.88、14.40、7.51、2.86和1.00,对各药剂的敏感性均差异显著。南宁和田阳种群对氯虫苯甲酰胺、溴氰菊酯、氟氯氰菊酯、甲氰菊酯和高效氯氰菊酯的敏感性差异均较大,相对毒力指数分别在1.00～41.88和1.00～79.25。两个种群对同种杀虫剂的敏感性也有所差异,除对高效氯氰菊酯的敏感性差异不显著外,对其余药剂的敏感性均差异显著。     

3种拟除虫菊酯类农药对草地贪夜蛾的毒力测定及田间应用效果评价

评价使用氟氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯喷雾和喇叭口点施处理对草地贪夜蛾的活性与防效,为草地贪夜蛾综合防控提供技术支持。采用喷雾法测定了3种农药原药对草地贪夜蛾3龄幼虫毒力;采用喷雾和喇叭口点施2种方式田间施用5.7%氟氯氰菊酯乳油、25 g/L溴氰菊酯乳油、5.0%高效氯氟氰菊酯乳油,药后第1天、第3天、第7天调查挂牌标记玉米上草地贪夜蛾活虫数,计算防治效果。3种拟除虫菊酯农药对草地贪夜蛾3龄幼虫LC 50值大小顺序依次为氟氯氰菊酯(29.80 mg/L)<高效氯氟氰菊酯(42.39 mg/L)<溴氰菊酯(49.88 mg/L);3种拟除虫菊酯类农药在54.00 g a.i./ha剂量下均能明显降低草地贪夜蛾田间虫口数量,同等剂量防效氟氯氰菊酯乳油>高效氯氟氰菊酯乳油>溴氰菊酯微乳剂。由于草地贪夜蛾低龄幼虫和高龄幼虫取食部位有差异,喷雾法防治低龄幼虫的效果好,喇叭口点施防治高龄幼虫的效果好。拟除虫菊酯类农药对草地贪夜蛾具有较好的防治效果,喇叭口点施方式节省药剂、提高对高龄幼虫的防效,在防治草地贪夜蛾等害虫方面具有广阔的应用前景。     

棉铃虫P450基因CYP9A12酵母表达产物对拟除虫菊酯的代谢作用

细胞色素P450氧化酶解毒代谢作用增强是棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂产生抗性的主要原因,棉铃虫细胞色素P450氧化酶基因CYP9A12的组成型过量表达与拟除虫菊酯抗性相关。为了进一步明确棉铃虫细胞色素P450氧化酶基因CYP9A12与拟除虫菊酯类杀虫剂抗性的关系,采用酿酒酵母Saccharomyces cerevisiae表达系统异源表达了CYP9A12基因,检测了该基因的酵母表达产物对溴氰菊酯、氟氯氰菊酯、甲氰菊酯和联苯菊酯4种药剂的离体代谢作用。结果表明：含有CYP9A12外源基因的重组酵母细胞裂解液对溴氰菊酯、氟氯氰菊酯和联苯菊酯的代谢率分别为8.58,5.85和3.94 pmol/min·mg protein,而没有检测到对甲氰菊酯的代谢。本研究表明了CYP9A12具有代谢多种拟除虫菊酯的能力,也为CYP9A12参与拟除虫菊酯的解毒代谢提供了直接证据。     

不同杀虫剂处理对赤拟谷盗P450基因诱导表达特性影响

害虫细胞色素P450基因可被杀虫剂迅速诱导,然而当前对不同杀虫剂处理下赤拟谷盗P450基因诱导表达特性的研究较少。本研究首先通过序列比对选取了来自不同家族的8个赤拟谷盗P450基因CYP4G7、CYP4Q4、CYP4BR3、CYP12H1、CYP6BK11、CYP9D4、CYP9Z5和CYP345A1,然后采用四种不同杀虫剂氯氰菊酯、氟氯氰菊酯、氯菊酯和吡虫啉对赤拟谷盗20 d幼虫进行生物测定,再根据生测结果以四个药剂亚致死剂量分别处理幼虫,并采用荧光定量PCR分析8个P450基因的表达特性。结果表明,CYP4G7和CYP345A1可以分别被氯氰菊酯(分别上调1.97倍和2.06倍)、氟氯氰菊酯(2.00倍和2.03倍)和氯菊酯(1.73倍和1.81倍)显著诱导,而CYP4BR3和CYP345A1可以被吡虫啉(分别上调1.99倍和1.93倍)显著诱导。本研究结果表明赤拟谷盗P450基因的显著诱导与基因家族类型以及农药品种有关。     

棉铃虫对氰戊菊酯-辛硫磷混剂的抗性演化及解毒酶活性变化

用氰戊菊酯-辛硫磷混剂（有效成分1∶10,简称氰-辛混剂）对棉铃虫Helicoverpa armigera室内品系(YS)进行16代的抗性选育,获得棉铃虫对氰-辛混剂的抗性品系（YS-FP）。YS-FP品系与YS品系相比,对氰-辛混剂的抗性为14.7倍,对其中的单剂氰戊菊酯和辛硫磷的抗性分别为2 170倍和3.1倍。随着筛选的进行,氰戊菊酯和辛硫磷之间的共毒系数在F₂代出现短暂的增加,然后逐渐降低,它们之间的互作由增效变为拮抗。交互抗性测定结果表明,YS-FP品系对氯氰菊酯、溴氰菊酯、三氟氯氰菊酯、三唑磷和灭多威产生了明显的交互抗性,对硫丹、多杀菌素和爱玛菌素没有产生交互抗性。YS-FP品系6龄幼虫中肠细胞色素P450氧化酶甲氧基香豆素O-脱甲基活性为YS品系的10倍,3龄幼虫谷胱甘肽S-转移酶和酯酶活性分别是YS品系的1.7倍（CDNB结合作用）和2.4倍（α-NA 酯酶水解作用）。氰-辛混剂的筛选导致了棉铃虫多种解毒酶活性的增加,特别是细胞色素P450氧化酶活性增强最为明显。本研究结果表明氰-辛混剂对棉铃虫的筛选导致了广谱的交互抗性和多种代谢抗性机理,并且两个单剂之间的互作由增效变为拮抗,因此氰 辛混剂在棉铃虫抗性治理中的作用是有限的和暂时的。     

Comparison of cytochrome P-450 levels in adult rat liver, postnatal rat liver, and primary cultures of postnatal rat hepatocytes

A system of primary cultures of postnatal rat hepatocytes has been developed to serve as an experimental model for drug metabolism and toxicity investigations. The purpose of this study was to examine the reported loss of cytochrome P-450 of hepatocytes when placed in culture and to compare activity in culture to intact liver and freshly isolated hepatocytes. A medium enriched with several hormones and a system of floating filters as a substratum for cell attachment were investigated as methods to reduce the expected loss of cytochrome P-450. When compared to initial values of cytochrome P-450 in whole liver and isolated hepatocytes, these methods failed to prevent the reduction of cytochrome P-450 in culture. However, our results compare favorably with other values reported in the literature.     

Structure-activity relationships for halobenzene induced cytotoxicity in rat and human hepatocytes

Halobenzenes are ubiquitous environmental contaminants, which are hepatotoxic in both rodents and humans. The molecular mechanism of halobenzene hepatotoxicity was investigated using Quantitative structure-activity relationships (QSAR) and accelerated cytotoxicity mechanism screening (ACMS) techniques in rat and human hepatocytes. The usefulness of isolated hepatocytes for prediciting in vivo xenobiotic toxicity was reassessed by correlating the LC(50) of 12 halobenzene congeners in phenobarbital (PB) induced rat hepatocytes in vitro determined by ACMS to the hepatotoxicities reported in vivo in PB-induced male Sprague-Dawely (SD) rats. A high correlation (r(2)=0.90) confirmed the application of hepatocytes as a "gold standard" for toxicity testing in vitro. QSARs were derived to determine the physico-chemcial variables that govern halobenzene toxicity in PB-induced rat, normal rat and human hepatocytes. We found that toxicity in normal rat and normal human hepatocytes both strongly correlate with hydrophobicity (logP), ease of oxidation (E(HOMO), energy of the highest molecular orbital) and on the asymmetric charge distribution according to arrangement of halogen substituents (dipole moment, mu). This suggests that halobenzene interaction with cytochrome P450 for oxidation is the metabolic activating path for toxicity and is similar in both species. In PB-induced rat hepatocytes the QSAR derivation is changed, where halobenzene toxicity strongly correlates to logP and dipole moment, but not E(HOMO). The changed QSAR suggests that oxidation is no longer the rate-limiting step in the cytotoxic mechanism when CYP2B/3A levels are increased, confirming CYP450 oxidation as the metabolic activating step under normal conditions.     

Hydroxylation products of hydrophobic xenobiotics--stabilizers of cytochrome P-450 in the hepatocytes

The effects of the hydroxylation product 3,4-benzo(a)pyrene and the free radical scavenger 1,2,3-trioxybenzene on cytochrome P-450 degradation in isolated rat hepatocytes induced by the Fe2+-ADP + NADPH system activating lipid peroxidation (LPO) were investigated. During incubation of hepatocytes, cytochrome P-450 is destroyed due to accumulation of LPO products. Addition of the free radical scavenger 1,2,3-trioxybenzene and the monoxygenase substrate 3,4-benzo(a)pyrene to the incubation medium induces inhibition of LPO and simultaneous stabilization of cytochrome P-450. Deceleration of malonic dialdehyde production by the free radical scavenger of the monoxygenase substrate suggests that both the compounds stabilize cytochrome P-450. It is assumed that in liver hepatocytes, exogenous free radical scavengers of the phenolic type and the products of their decarboxylation protect cytochrome P-450 against the LPO-induced destruction via oxidative metabolism of hydrophobic substrates.     

Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes

Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P-450 (P-450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P-450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P-450IA subfamily, and clofibrate the P-450-IVA subfamily. In cocultures, after 6 days, most P-450 enzymes were still expressed while P-450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell-cell interactions, that it is possible to maintain reasonable xenobiotic-metabolizing-enzyme expression; however, these conditions have to be improved in order to preserve better P-450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.     

Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone.

The loss of cytochrome P-450 in cultured rat hepatocytes can be prevented by substituted pyridines, especially isonicotinamide, 3-hydroxypyridine and metyrapone. The effect of these compounds is independent of protein synthesis, suggesting that they maintain pre-existing cytochrome P-450. The efficiency of pyridines at maintaining cytochrome P-450 in hepatocyte culture is highly correlated with their ability to bind to this cytochrome, suggesting that ligand formation with cytochrome P-450 prevents its accelerated turnover in liver cell culture.     

Inhibition of protein synthesis: a basis for tunicamycin-induced decrease in rat liver cytochrome P-450

Tunicamycin caused a dose and time dependent decrease in cytochrome P-450 in rat liver. A dose of 50 micrograms/kg caused a decrease of about 50% in 72 hours. A similar decrease in the activities of rat liver microsomal aniline hydroxylase, aminopyrine N-demethylase and ethoxycoumarin O-deethylase were also seen after the tunicamycin treatment. Tunicamycin also suppressed food and water intake but the decrease in cytochrome P-450 was not related to these effects. NADPH cytochrome c reductase was not markedly decreased by tunicamycin. A decrease in cytochrome P-450 was also observed in cultured rat hepatocytes treated with tunicamycin. It decreased incorporation of [35S]-methionine into total proteins as well as into various cytochrome P-450 isozymes of rat hepatocytes. This indicates that a decrease in protein synthesis may be responsible for the tunicamycin-induced decrease in cytochrome P-450 and drug metabolism.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Role of cytochrome P450 3A in the metabolism of mefloquine in human and animal hepatocytes

We studied mefloquine metabolism in cells and microsomes isolated from human and animal (monkey, dog, rat) livers. In both hepatocytes and microsomes, mefloquine underwent conversion to two major metabolites, carboxymefloquine and hydroxymefloquine. In human cells and microsomes these metabolites only were formed, as already demonstrated in vivo, while in other species several unidentified metabolites were also detected. After a 48 hr incubation with human and rat hepatocytes, metabolites accounted for 55-65% of the initial drug concentration, whereas in monkey and dog hepatocytes, mefloquine was entirely metabolized after 15 and 39 hrs, respectively. The consumption of mefloquine was less extensive in microsomes, and unchanged drug represented 60% (monkey) to 85-100% (human, dog, rat) of the total radioactivity after 5 hr incubations. The involvement of the cytochrome P450 3A subfamily in mefloquine biotransformation was suggested by several lines of evidence. Firstly, mefloquine metabolism was strongly increased in hepatic microsomes from dexamethasone-pretreated rats, and also in human and rat hepatocytes after prior treatment with a cytochrome P450 3A inducer. Secondly, mefloquine biotransformation in rifampycin-induced human hepatocytes was inhibited in a concentration-dependent manner by the cytochrome P450 3A inhibitor ketoconazole and thirdly, a strong correlation was found between erythromycin-N-demethylase activity (mediated by cytochrome P450 3A) and mefloquine metabolism in human microsomes (r=0.81, P < 0.05, N=13). Collectively, these findings concerning the role of cytochrome P450 3A in mefloquine metabolism may have important in vivo consequences especially with regard to the choice of agents used in multidrug antimalarial regimens.     

Human cytochrome P-450 PB-1: A multigene family involved in mephenytoin and steroid oxidations that maps to chromosome 10

The cytochrome P-450 monooxygenase system possesses catalytic activity toward many exogenous compounds (e.g., drugs, insecticides, and polycyclic aromatic hydrocarbons) and endogenous compounds (e.g., steroids, fatty acids, and prostaglandins). Multiple forms of cytochrome P-450 with different substrate specificities have been isolated. In the present paper we report the isolation and sequence of a cDNA clone for the human hepatic cytochrome P-450 responsible for mephenytoin (an anticonvulsant) oxidation. The mephenytoin cytochrome P-450 is analogous to the rat cytochrome P-450 form termed PB-1 (family P450C2C). We also report that human PB-1 is encoded by one of a small family of related genes all of which map to human chromosome 10q24.1-10q24.3. The endogenous role of this enzyme appears to be in steroid oxidations. This cytochrome P-450 family does not correspond to any of the hepatic cytochrome P-450 gene families previously mapped in humans.     

Apparent maintenance of cytochrome P 450 by nicotinamide in primary cultures of rat hepatocytes.

The sole addition of a high, unphysiological, concentration of nicotinamide (25 mM) to a cell culture medium was found to maintain the cytochrome P 450 concentration of rat hepatocytes cultured for 24 hours at 71% of the level found in intact liver, whilst hepatocytes cultured without nicotinamide contained only 20% of their initial cytochrome P 450. Furthermore the P 450 concentration of hepatocytes cultured for 24 hours in the presence of 25 mM nicotinamide could be increased to the same level as found in intact rat liver by the inclusion of 1 mM nicotinamide into the medium used for cell isolation. Although the mechanism of action of nicotinamide is unknown this simple system for the maintenance of cytochrome P 450 in hepatocyte culture could provide the opportunity to study, under defined conditions $\text{in vitro}$, the factors that regulate cytochrome P 450 and hence determine hepatotoxicity and hepatocarcinogenesis.