INDUCTION OF CYTOCHROME P-450 1A1 IN HUMAN HEPATOMA HepG2 AND LUNG CARCINOMA NCI-H322 CELLS BY MOTORCYCLE EXHAUST PARTICULATE

A dose-response study was conducted to examine the growth suppression associated with developmental lead exposure in a rat model and to determine the endocrine mechanisms underlying these effects. Ad libitum intake of lead acetate (0.05% to 0.45% w/v) was initiated in time-impregnated female Sprague-Dawley rats (n = 10-15/ group) at gestational day 5. At birth, pups were culled to four male and four females per litter. Lead exposure of dams continued until weaning, following which lead exposure of pups was continued until sacrifice at age 21, 35, 55, and 85 days. Birth weight and prepubertal and pubertal growth rates were significantly suppressed. Growth rates were suppressed to a much greater degree in male as compared to female pups. Decreased growth rates were accompanied by a significant decrease in plasma insulin-like growth factor 1 (IGF1) concentrations and (1) a significant increase in pituitary growth hormone (GH) content during puberty in pups of both sexes, (2) a delay in the developmental profiles of the GH-dependent male-specific liver enzymes cytochrome P-450 CYP2C11 and N -hydroxy-2-acetylaminofluorene sulfotransferase, and (3) continued suppression of these enzymes in lead-exposed adult male pups. In addition, significant decreases in plasma sex steroids, testosterone (male) and 17 -estradiol (female), were observed during puberty. Postpuberty, at age 85 d, both IGF1 and sex steroid levels were indistinguishable from control pups despite continued lead exposure. Growth rates were also similar in control and lead-exposed pups between age 57 and 85 d. Data suggest that the mechanism underlying lead-induced sex-independent suppression of growth observed in these studies involves disruption of GH secretion during puberty. It is possible that the mechanisms underlying the greater suppression of somatic growth observed at puberty in lead-exposed male offspring may be due to the additional hypoandrogenization produced by the action of lead on the hypothalamic-pituitary-testicular axis.     

CHARACTERIZATION OF CYTOCHROME P450-DEPENDENT AND GLUTATHIONE TRANSFERASE ACTIVITIES IN SV40-IMMORTALIZED UROEPITHELIAL CELL LINES: POSSIBLE ROLE IN TRANSFORMATION AND TUMOR FORMATION

An in vitro/in vivo transformation system has been developed as a model for bladder tumorigenesis. SV40-immortalized human uroepithelial cells are exposed to putative carcinogens and then implanted into athymic nude mice to testfortumorigenesis.Studieswith4-aminobiphenyl(4-ABP)demonstratedthat one cell line, SV-HUC-PC, was sensitive to chemical-induced transformation and another line, SV-HUC-BC, was refractory.Weare currently testing this system as a model to identify occupational carcinogens and develop biomarkers of exposure and effects of exposure. As part of this study, we examined P450- dependent metabolism, glutathione transferase, and the effects of chemicals on deoxyribonucleic acid(DNA)synthesisandrepair inSV-HUC-PC andSV-HUCBC. Activities for CYP1A1/1A2, CYP3A, and CYP2B1/2B2 were estimated by determining o -dealkylation of ethoxy-, benzoxy-, and pentoxy-resorufin, respectively. Coumarin hydroxylase and p -nitrophenol hydroxylase were used to estimate CYP2A and CYP2E1, respectively. SV-HUC-PC microsomes had fivefold greaterCYP1A1/1A2activityandtwofoldhigherCYP3AactivitythanSV-HUCBC. CYP2B1/2B2 and CYP2A activities and glutathione transferase were not different between the two cell lines. DNA synthesis and repair, by BrdU incorporation, was not different between the two lines when N-methyl-N-nitroN-nitrosoguanidine (MNNG) or other reactive metabolites were tested; however, SV-HUC-PC was more sensitive to n -nitrosodimethylamine, 4-ABP, and 4,4-methylene bis (2-chloroaniline) (MOCA). The data demonstrate that, while these cells have retained form-specific P450 activities, SV-HUC-PC has greater CYP1A1/1A2 and CYP3A activities.     

CHARACTERIZATION OF SOLUBILIZED CYTOCHROME P-450

Porcine liver microsomes were subjected to digitonin, n-butanol and sonication treatment to solubilize cytochrome P-450 and other mixed-function oxidation components. The best preparation achieved with 2% digitonin gave characteristic P-450, CN-and NO₂- difference spectra. It was further purified by ammonium sulfate fractionation and column chromatography. Column chromatography resulted in the separation of cytochrome P-450 subparticles from cytochrome P-420 subparticles; both contained negligible quantities of cytochrome b₅ and significant amounts of ferricyanide reductase and NADPH oxidase activity.     

INTERACTIONS BETWEEN CYTOCHROME P-450 ACTIVITIES AND OZONE-INDUCED MODULATORY EFFECTS ON ENDOTHELIAL PERMEABILITY IN RABBIT LUNGS: Influence of Gender

The effects of rabbit exposure to ozone (O3) (0.4 ppm for 4 h) on two different cytochrome P-450 (CYP450)-dependent activities were investigated. In turn, the role of CYP450 in the inhibitory effect of O 3 on acetylcholine (ACh)-evoked increase in endothelial permeability was also assessed. Immediately after the period of exposure, rabbits of both sexes were sacrificed and their lungs were extracted. Some lungs were used for preparation of microsomes and measurement of 7-ethoxyresorufin O -deethylase (EROD) and parathion oxidase activities. Other rabbit lungs were isolated and recirculatingly blood-free perfused. Arterial, venous pressures, and lung weight were continuously recorded. Capillary pressure was measured by applying the double occlusion method. Capillary filtration coefficient (K f,c) was evaluated by measuring the amount of fluid filtering through the endothelium when vascular pressures were suddenly increased. Dose-response curves to ACh were constructed in air- or O3-exposed rabbits. Some animals were pretreated with piperonyl butoxide (PBO), a well-known inhibitor of CYP450. O3 significantly reduced both EROD and parathion oxidase lung microsomal activities in females, while it had no effect in males. Exposure to O3 strongly inhibited the ACh-induced increase in K f,c. Pretreatment with PBO reversed the modulatory effect of O 3 on endothelial permeability in male rabbits, but not in females. It was concluded (1) that inhibition of 2 different CYP450-dependent activities after exposure to 0.4 ppm O3 for 4 h appears to be a gender-dependent phenomenon, and (2) that CYP450 is probably involved in the O 3-evoked inhibitory mechanism against ACh-induced increase in endothelial permeability, but only in males.     

EFFECTS OF GOLD SODIUM THIOMALATE ON CYTOSOLIC COPPER AND ZINC IN THE RAT KIDNEY AND LIVER TISSUES

1. Male Wistar rats were given a single subcutaneous injection of gold sodium thiomalate and killed 7 days later. The binding of Au, Zn and Cu to the kidney and liver cytosolic proteins of control and gold-treated rats was determined. 2. In the renal cytosol of Au(I)- exposed rats, the binding of Cu to the low-molecular-weight (LMW) proteins increased by 62%, and to the high-molecular-weight (HMW) proteins the binding decreased by 54%. The incorporation of Cu into the liver cytosolic proteins increased, in both, the HMW and the LMW proteins. The binding of Zn into the renal cytosolic proteins was increased by 39% (HMW proteins) and 100% (LMW proteins). Au(I) had little effect on the binding of Zn to the cytosolic proteins in the liver. 3. It is suggested that the therapeutic action of gold complexes may be mediated, to some extent, by its effects on the metabolism of Cu and/or Zn.     

黄芩苷对小鼠肝细胞色素P450的选择性诱导

目的　观察黄芩苷对小鼠肝细胞色素P450及其亚家族的影响。方法　用紫外分光光度法分别测定小鼠肝微粒体细胞色素P450与b5含量及氨基比林N-脱甲基酶(ADM)、7-乙氧基香豆素O-脱乙基酶(ECD)、苯并芘羟化酶(AHH)活性。用蛋白印迹杂交技术鉴定细胞色素P450同功酶。结果　黄芩苷可使小鼠肝微粒体细胞色素P450含量显著增加,并使ADM,ECD及AHH 3种酶活力显著增强。对6种P450同功酶的鉴定结果显示,黄芩苷可选择性诱导1A1,2B1及2C11 3种同功酶,对细胞色素b5含量及3A2,2D1和2E1 3种同功酶无诱导作用。结论　黄芩苷对小鼠肝细胞色素P450有选择性诱导作用。     

METABOLISM AND CYTOTOXICITY OF AFLATOXIN B 1 IN CYTOCHROME P-450-EXPRESSING HUMAN LUNG CELLS

The mycotoxin aflatoxin B 1 (AFB 1 ) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB 1 has been detected in dusts generated in the processing and transportation of AFB 1 -contaminated products. Inhalation of grain dusts contaminated with AFB 1 may be a risk factor in human lung cancer. Aflatoxin B 1 requires cytochrome P-450 (CYP)-mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB 1 activation in human lung is not well understood. To investigate whether these CYPs can activate AFB 1 at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells (BEAS-2B) that were transfected with cDNA for CYPs 3A4 (B3A4) or 1A2 (B-CMV1A2) were used. B-CMV1A2 cultured in 15 n M AFB 1 produced the AFB 1 -glutathione conjugate (AFB 1 -GSH) and aflatoxin M 1 (AFM 1 ), while B3A4 cells produced only aflatoxin Q 1 (AFQ 1 ) at 0.15 M AFB 1 . Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 m M AFB 1 . Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB 1 to AFB 1 8,9-epoxide (AFBO), while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S -transferase (GST)-mediated trapping of enzymatically generated AFB 1 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB 1 compared to B3A4 cells, and were 6000-fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB 1 , followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB 1 primarily at higher AFB 1 concentrations, was also responsible for significant AFB 1 toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB 1 , even at environmentally relevant concentrations.     

五味子乙素对小鼠肝细胞微粒体细胞色素P-450的诱导作用

五味子乙素系从中药五味子分离出的成分之一,有抗肝损伤和解毒作用,同时亦引起小鼠肝脏重量增大。经进一步研究,发现五味子乙素对正常小鼠肝脏每克组织中水分、蛋白质、RNA、糖原及总脂含量均无明显影响,但在整个肝脏中上述成分含量均显著增加。每克肝组织中DNA含量稍降低,整个肝脏中DNA含量则无明显变化。对部分切除肝脏的小鼠再生肝,五味子乙素能明显引起整个肝脏中蛋白质,PLNA和DNA含量以及细胞核分裂数增加。此外,五味子乙素能显著促进¹⁴C-苯丙氨酸掺入肝脏蛋白质,并使肝细胞微粒体细胞色素P-450及蛋白质含量显著增加。以上结果表明,五味子乙素对药酶有诱导作用,其引起的肝脏增大主要系肝实质细胞体积增大,同时伴有肝细胞增生。五味子乙素引起的肝脏增大似非病理性损害的表现,而是由于诱导药酶同时出现肝细胞肥大的表现。     

METABOLISM OF N-NITROSOBENZYLMETHYLAMINE BY HUMAN CYTOCHROME P-450 ENZYMES

N-Nitrosobenzylmethylamine (NBzMA) is a potent esophageal carcinogen in rodents, and has been found as a dietary contaminant in certain areas of China where esophageal cancer is endemic. To determine which cytochrome P-450 enzymes in humans are primarily responsible for NBzMA metabolism, microsomes from lymphoblastoid cell lines expressing a panel of human cytochrome P-450s (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4) and a panel of 10 different human liver microsomal preparations were examined for their abilities to metabolize [³H]NBzMA. In addition, the ability of human liver microsomes to form various NBzMA metabolites was correlated with the abilities of these preparations to metabolize coumarin, ethoxyresorufin, chlorzoxazone, 7-ethoxy-4-trifluoromethylcoumarin, S-mephenytoin, and nifedipine. NBzMA metabolites were quantitated by reversed-phase high-performance liquid chromatography (HPLC) coupled with flow-through radioactivity detection. Major metabolites included benzaldehyde, benzyl alcohol, benzoic acid, and several uncharacterized radioactive peaks. Of the representative P-450 activities, only CYP2E1 and CYP2A6 catalyzed substantial metabolism of NBzMA. Compared to CYP2E1, CYP2A6 metabolized NBzMA more readily. NBzMA acted as a potent inhibitor of coumarin 7-hydroxylation in CYP2A6 microsomes. Human liver microsomes metabolized NBzMA readily. NBzMA metabolite formation was most highly correlated with coumarin 7-hydroxylase activity, a marker of CYP2A6 activity. 8-Methoxypsoralen substantially inhibited NBzMA metabolism in human hepatic microsomes. When the effects of the potent isothiocyanates PEITC and PHITC were analyzed on microsomes from cell lines expressing CYP2E1 and CYP2A6, it was found that PEITC inhibited both enzymes, PHITC was the more effective inhibitor of CYP2E1, and PHITC was an ineffective inhibitor of CYP2A6. Collectively, these data indicate that CYP2A6 and, to a lesser degree, CYP2E1 are important P-450 enzymes in the activation of NBzMA in human systems.     

氟他胺在大鼠肝微粒体经细胞色素P450 1A2代谢的性别差异

目的体外研究大鼠肝微粒体细胞色素P450 1A2(CYP1A2)对氟他胺(flutamide Flu)代谢的性别差异影响。方法制备正常♀♂大鼠肝微粒体,用CYP1A2抗体与氟他胺(2 mg·L^-1)共同温孵,测定氟他胺主要代谢产物2-羟基氟他胺(2-hydroxyflutamide, HF)和原药的浓度比(HF/Flu),评价氟他胺在大鼠肝微粒体代谢的性别差异。结果在CYP1A2抗体浓度为1∶400,孵育时间为30 min条件下,氟他胺在♂大鼠肝微粒体中的HF/Flu为(1.5±0.6),而♀动物为(0.9±0.4)。不同性别大鼠肝微粒体对氟他胺的代谢存在性别差异(P<0.01)。结论Flu在♂大鼠肝微粒体中代谢快,而在♀大鼠肝微粒体中代谢较慢。♂大鼠体内的CYP1A2酶活性高于♀大鼠。     

氨基酸和酵母膏对谷胱甘肽发酵的影响

研究了氨基酸和酵母膏对酵母发酵生产谷胱甘肽（ＧＳＨ）的影响。结果表明，多种氨基酸能促进酵母的生长，对ＧＳＨ的生物合成却没有明显促进作用，而半胱氨酸则表现为在抑制酵母生长的同时显著提高胞内ＧＳＨ含量。半胱氨酸初始浓度为５ｍｍｏｌ／Ｌ时ＧＳＨ比产生速率最大，为３．７ｍｇ·ｇｃｅｌ－１·ｈ－１。酵母膏对酵母的生长和ＧＳＨ的合成均有显著的促进作用。若不添加酵母膏，则３％糖浓度下胞内ＧＳＨ含量只有１％糖浓度下的５０％；而若在３％和１％糖浓度下分别添加０．６％的酵母膏，前者胞内ＧＳＨ含量比不加酵母膏的对照提高了一倍左右；３％糖浓度（补糖操作）发酵液中ＧＳＨ总量（９２．５ｍｇ／Ｌ）比１％糖浓度的ＧＳＨ总量高４８％。     

中药戒得安对吗啡依赖大鼠血清和尿中吗啡含量的影响

目的··:考察戒得安的戒毒机理。方法··:应用放射免疫法测定吗啡依赖大鼠血清和尿液中吗啡含量。结果·· :戒得安能显著增加依赖大鼠在停用吗啡后尿液及血清中吗啡的含量。结论··:戒得安具有加速体内毒品排出体外的速度,而达到脱毒的疗效     

INHIBITION OF AGONIST-INDUCED VASOCONTRACTION AND IMPAIRMENT OF ENDOTHELIUM-DEPENDENT VASORELAXATION BY EXTRACT OF MOTORCYCLE EXHAUST PARTICLES IN VITRO

The in vitro effects of motorcycle exhaust particulate extract (MEPE) on blood vessels were studied in thoracic aorta isolated from Wistar rat. The MEPE relaxed the phenylephrine-precontracted aorta with an EC50 value of 0.05 +/- 0.004 mg/ ml. This relaxing effect of MEPE persisted in endothelium-denuded aorta, suggesting that the relaxation induced by MEPE is endothelium-independent. The phenylephrine-induced vasocontraction and inositol 1,4,5-triphosphate formation were inhibited concentration dependently in aorta pretreated with MEPE. However, the high-K+-induced vasocontraction and the Ca2+ sensitivity of the contractile proteins were not significantly affected by MEPE. In addition to the inhibitory effects on agonist-induced contraction, the vasorelaxing effects both of acetylcholine and of sodium nitroprusside were impaired by MEPE. The inhibitory effects of MEPE on acetylcholine and sodium nitroprusside, but not phenylephrine, were reversed by cotreatment with superoxide dismutase. These results showed that the MEPE, added in vitro, inhibited the phenylephrine-induced, but not depolarization-induced, vasocontraction of aorta. The MEPE also impaired the vasorelaxation induced by acetylcholine in a superoxide anion-dependent manner.     

ALTERATIONS OF MOTOR NERVE FUNCTIONS IN ANIMALS EXPOSED TO MOTORCYCLE EXHAUST

The effect of motorcycle exhaust (ME) on the motor nerve was studied using animals exposed to the exhaust by inhalation, intratracheal, or intraperitoneal administration of ME particulate (MEP). A 4-wk ME inhalation and intratracheal instillation of MEP for 1 d in rats or intraperitoneal administration of MEP (0.5 g/kg/d for 1 d and 0.1 g/kg/d for 2 d) in mice significantly decreased both rota-rod performance and motor nerve conduction velocity. The effect of some polycyclic aromatic hydrocarbons on the motor nerve was also investigated. Treatment with benzo[a]pyrene (0.05 and 0.1 g/kg/d for 3 d), but not pyrene (0.1 g/kg/d for 3 d), resulted in significant decrease of motor nerve conduction velocity in mice. Moreover, the Na + ,K + -ATPase activities of sciatic nerves isolated from ME-, MEP-, or benzo[a]pyrene-exposed animals were decreased. Treatment with pyrene did not markedly affect the Na + ,K + -ATPase activity of sciatic nerve. The rats exposed to ME for 4 wk showed increases in blood and sciatic nerve manganese levels. Results indicate that motorcycle exhaust produces adverse effects on the motor nerve, which is associated with a fall in nerve Na + ,K + -ATPase activity.     

槲皮素对体外培养的SD大鼠破骨细胞增殖及凋亡的影响

目的　研究槲皮素对体外培养的SD大鼠破骨细胞增殖及凋亡的影响。方法　体外机械分离SD大鼠破骨细胞 ,在空白对照及含槲皮素 2 .5、10、4 0mmol·L-1的 4种MEM培养基中进行培养 ,并于 4 8、72、96、12 0h涂片经瑞氏染色和TUNEL法染色观察各培养孔内破骨细胞数目、形态、凋亡情况。结果　各组破骨细胞数目随着药物浓度增加而减少 ,P <0 .0 0 5。药物浓度的增加使破骨细胞凋亡的比例呈增加趋势。结论　槲皮素能够明显抑制大鼠体外培养的破骨细胞增殖 ,并存在诱导其凋亡的作用。