茶多酚对氯霉素毒性作用的影响

氯霉素是一种广谱抗菌素，它的代谢失活主要器官为肝脏，首先被肝脏中细胞色素Ｐ４５０（ＣＹＰ４５０）单加氧酶氧化。我们实验室曾证明β－萘黄酮（β－ＮＦ）可诱导人羊膜上皮细胞ＦＬ系（ＦＬ细胞）的ＣＹＰＰ４５０１Ａ１同功酶的活性，而茶多酚可抑制ＦＬ细胞ＣＹＰ４５０１Ａ１同功酶的活性。本实验以人羊膜上皮细胞ＦＬ系（ＦＬ细胞）和转入ＣＹＰ４５０１Ａ１基因的ＦＬ细胞（ＦＬ－１Ａ１细胞）为实验材料，观察茶多酚（Ｇ     

血清F对AFP阴性或低浓度原发性肝癌的诊断分析

为了探讨血清Ｆ对ＡＦＰ不同浓度的原发性肝癌的诊断意义，自１９９３年１月至１９９４年１２月，对２４３例原发性肝癌和４６例健康对照组进行了血清ＡＦＰ、Ｆ的检测。结果显示：原发性肝癌的血清ＡＦＰ（〉４００μｇ／Ｌ）、Ｆ（男〉２５０μｇ／Ｌ、女〉１５０μｇ／Ｌ）的阳性率分别为４７．７％、７２．４％，与对照组比较二者差异均有高度显著性（Ｐ均〉０．０１）。在ＡＦＰ阴性、低浓度和阳性中Ｆ阳性率分别为７３．２％（     

TAM和MPA对人卵巢癌细胞凋亡和PCNA、EGFR表达的影响

探讨三苯氧胺（ｔａｍｏｘｉｆｅｎ，ＴＡＭ）和甲孕酮（ｍｅｄｒｏｘｙｐｒｏｇｅｓｔｅｒｏｎｅ ａｃｅｔａｔｅ，ＭＰＡ）对人卵巢癌细胞增殖和凋亡的影响。方法：用不同浓度（０．１μｍｏｌ／Ｌ、１μｍｏｌ／Ｌ、１０μＭＯＬ／ｌ）的ＴＡＭ和ＭＰＡ作用于人卵巢癌纱３Ａ０，分别体外培养４８ｈ，７２ｈ，９６ｈ。用台盼蓝活细胞拒染法动态观察活细胞数，免疫组化法检测增殖的（ｐｒｏｌｉｆｅｒａｔｉｏｎ ｃｅｌｌ ｎｕｃ     

非小细胞肺癌化学治疗与血清sAPO-1/Fas、NO的变化

目的 观察化疗药物替尼泊甙（ＶＭ－２６）、表阿霉素（ＥＰＩ）、顺铂（ＤＤＰ）治疗非小细胞肺癌的疗效、毒性及治疗前后血清ｓＡＰＯ－１／Ｆａｓ、ＮＯ含量的变化。方法４２例诊断明确的晚期非小细胞肺癌（ＮＳＣＬＣ）患者（鳞癌２８例,腺癌１２例,未分化癌２例）采用ＶＭ－２６、ＥＰＩ、ＤＤＰ联合化疗２个周期。化疗前后分别用比抗体夹心ＥＬＩＳＡ方法测定血清ｓＡＰＯ－１／Ｆａｓ含量和用酶还原法测定血清ＮＯ含量。结     

ELF与FAM方案治疗晚期胃癌57例临床分析

我科自１９８５年５月至１９９５年３月，用ＥＬＦ及ＦＡＭ方案治疗晚期胃癌５７例，其中ＥＬＦ组２５例，ＦＡＭ组３２例。疗效分别为５６％（１４／２５）和３１．２５％（１０／３２），完全缓解率为１２％（３／２５）和１２．５％（４／３２）。恶心呕吐的Ⅲ级毒性发生率：ＥＬＦ组为０％，ＦＡＭ组为２５％（８／３２）；口腔粘膜Ⅱ级以上毒性发生率：ＥＬＦ组为４４％（１１／２５），ＦＡＭ组为０％；ＥＬＦ组未见心脏毒性，而ＦＡＭ为１２．５％。两者疗效无统计学差异（Ｐ＞０．０１）。恶心呕吐发生率ＦＡＭ方案明显大于ＥＬＦ方案（Ｐ＜０．０２５），而ＥＬＦ组的口腔炎发生率明显高于ＦＡＭ组（Ｐ＜０．００１）。结果表明ＥＬＦ方案更适合于不能耐受强烈化疗或存在基础心脏疾病的进展期胃癌患者。     

卵巢癌细胞系BRCA1蛋白表达的雌激素调控

目的：观察不同浓度雌激素对卵巢癌细胞系乳腺癌／卵巢癌易感基因（ＢＲＣＡ１）蛋白表达的影响。方法：采用免疫组化及计算机图象分析方法，检测体外不同浓度（１０＾－９ｍｏｌ／Ｌ￣１０＾－６ｍｏｌ／Ｌ）的１７－β雌二醇（Ｅ２）对卵巢癌ＯＶＣＡＲ３和ＳＫＯＶ３细胞系ＢＲＣＡ１蛋白表达的影响。结论：雌激素能够诱导卵巢癌ＯＶＣＡＲ３和ＳＫＯＶ３细胞系ＭＲＣＡ１蛋白的表达，可通过该方法探索治疗卵巢癌的新途径。     

三氟拉嗪逆转肺癌细胞多药耐药性研究

目的研究三氟拉嗪（ＴＦＰ）逆转肺癌细胞多药耐药的作用。方法应用ＭＴＴ比色法体外实验研究三氟拉唪对多药耐药性人肺癌细胞株Ａ５４９／阿霉素（ＡＤＭ）耐药性的逆转作用。结果（１）ＴＦＰ对细胞具有药物浓度依赖性毒性作用，ＩＰＰＣ浓度的ＴＦＰ细胞抑制率可达２９．７％±８．７％。（２）ＴＦＰ可显著逆转细胞对顺铂、长春新碱、阿霉素的耐药性，与单独化疗药物比较，有显著差异（Ｐ＜０．０５或Ｐ＜０．０１）。其作用同样具有药物浓度依赖性，各药物浓度间细胞抑制率有显著差异（Ｐ＜０．０５或Ｐ＜０．０１）。结论三氟拉嗪具有逆转肺癌细胞耐药的作用。     

氧化酚砷对血液肿瘤细胞系的体外效应

目的：了解氧化酚砷（ｐｈｅｎｙｌａｒｓｉｎｅ ｏｘｉｄｅ,ＰＡＯ）对血液肿瘤细胞系的影响。方法：８种血液肿瘤系经０．０１ｕｍｏｌ／Ｌ、０．０５ｕｍｏｌ／Ｌ和０．１ｕｍｏｌ／Ｌ的ＰＡＯ处理达一定时间后,经台盼蓝排除法计数细胞活力的应用流式细胞仪检测细胞ＤＮＡ含量分布。结果和结论：在生长抑制和诱导凋亡方面,ＰＡＯ对血液肿瘤细胞的效庆谱相对较广,０．０５ｕｍｏｌ／Ｌ和０．１ｕｍｏｌ／Ｌ的ＰＡＯ能抑制某些     

半乳糖基修饰的反义硫代寡核苷酸对肝癌多药耐药细胞株MDR_1基因过度表达的抑制作用

设计合成三种互补ＭＤＲ１ｍＲＮＡ的反义硫代寡核苷酸（ＡＳＯＤＮ），分别互补ＭＤＲ１ｍＲＮＡ序列起始区、含ＡＵＧ起始密码子区及针对Ｌｏｏｐ形成位点区的序列，作用于阿霉素（ＤＯＸ）诱导的肝癌多药耐药细胞株ＢＥＬ－７４０２／ＤＯＸ。经流式细胞分析及ＲＴ－ＰＣＲ的方法检测，发现三种ＡＳＯＤＮ均能不同程度地降低ＢＥＬ－７４０２／ＤＯＸ细胞膜表面Ｐ－ＧＰ含量；同时ＭＤＲ１ｍＲＮＡ的表达也有轻度降低，其中尤以互补ＭＤＲ１ｍＲ－ＮＡＬｏｏｐ形成位点的ＡＳＯＤＮ抑制作用最强。以导向配体Ｇａｌ１０－ＰＬＬ修饰此ＡＳＯＤＮ后，作用于ＢＥＬ－７４０２／ＤＯＸ细胞，发现与相同剂量未修饰ＡＳＯＤＮ比较，Ｐ－ＧＰ含量由７６．８％降至１９．８％；对ＡＤＭ的ＩＣ５０由１．２８μｇ／ｍｌ降至０．４２μｇ／ｍｌ。实验说明，半乳糖基修饰的互补ＭＤＲ１ｍＲＮＡＬｏｏｐ形成位点的ＡＳＯＤＮ能够明显降低肝癌多药耐药细胞膜表面Ｐ－ＧＰ的含量，并较大程度地逆转多药耐药细胞ＢＥＬ－７４０２／ＤＯＸ对化疗药物的耐受性。     

HBV感染对原发性肝癌患者血清AFP值的影响

本文报道测定１０８例ＰＨＣ者的血清ＡＦＰ值和ＨＢＶ感染标记。７１例（６８．５％）ＡＦＰ值〉２０μｇ／Ｌ，其中６５例ＡＦＰ值〉１００μｇ／Ｌ；ＨＢｓＡｇ阳性者９６例（８８．９％），ＨＢｓＡｇ阴性者１２例（１１．１％），ＨＢｓＡｇ阳性者ＡＦＰ值的中位数为５０５μｇ／Ｌ（２０～２２３４μｇ／Ｌ）显著高于ＨＢｓＡｇ阴性者的２０μｇ／Ｌ（２０～３８３μｇ／Ｌ），ＨＢｓＡｇ阳性者ＡＦＰ值升高的峰值年龄组与ＰＨ     

Activation of trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene to genotoxic metabolites by rat and human cytochromes P450

In order to address the hypothesis that 6-aminochrysene (6-AC)is converted to genotoxic products by cytochrome P450 enzymesvia two activation pathways (N-hydroxylation and epoxidation),the activation of 6-AC and trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene(6-AC-diol) to genotoxic metabolites was examined in rat andhuman liver microsomal cytochrome P450 enzymes using Salmonellatyphimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009)as tester strains. The latter bacteria, an O-acetyl-transferase-overexpressingstrain, was highly sensitive to metabolites derived from activationof 6-AC, but not those from 6-AC-diol, using liver microsomesfrom phenobarbital-treated rats or a reconstituted monooxygenasesystem containing P4502B1 or -2B2, thus suggesting the rolesof P450 and acetyltransferase systems in the activation process.6-AC-diol, on the other hand, was activated very efficientlyby liver microsomes prepared from ß-naphthoflavone-treatedrats or a reconstituted system containing P4501A1 or -1A2; theactivation reaction is considered to proceed through diol-epoxideformation. The contribution of rat P4501A enzymes towards activationof 6-AC-diol was confirmed by the inhibitory effects on theactivation process of -naphthoflavone, a specific inhibitorof P4501 A-related activities, and antibodies raised againstpurified P4501A1 and -1A2. In humans, P4501A2 was found to bethe major enzyme involved in the activation of 6-AC-diol togenotoxic metabolites while the parent compound 6-AC was activatedmainly by P4503A4. Experiments using recombinant P450 proteinsexpressed in human lymphoblastoid cells lines showed that humanP4501A1 could also activate 6-AC-diol to reactive metabolitesat almost the same rate measured with P4501A2. In addition,P4502B6 was found to efficiently catalyze the activation of6-AC to genotoxic metabolites, and P4503A4 was active in theactivation of 6-AC-diol as well as 6-AC. Addition of purifiedrat epoxide hydrolase to the incubation mixture containing purifiedrat P4501A1 or microsomes expressing human P4501A1 caused inhibitionof activation of 6-AC-diol. These results suggest the existenceof different enzymatic activation pathways for 6-AC and 6-AC-diol.The former carcinogen may be N-hydroxylated principally by P4502Benzymes in rats and P4503A4 and -2B6 in humans and activationto its ultimate metabolites may proceed through esterificationof the N-hydroxy metabolites by an N-acetyltransferase. The6-AC-diol is metabolized to its ultimate diolepoxide productby P4501A enzymes in rat and human liver microsomes. P4503A4(humans) and P4503A2 (rats) may also contribute to some extentin the activation of 6-AC-diol, albeit at lower rates than thoseof P4501A enzymes.     

A new selective and potent inhibitor of human cytochrome P450 1B1 and its application to antimutagenesis.

Human cytochrome P450 (P450) 1B1 is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17beta-estradiol (E(2)) to 4-hydroxy E(2) by P450 1B1 has been postulated to be a factor in mammary carcinogenesis. The inhibition of recombinant human P450 1B1 by 2,4,3',5'-tetramethoxystilbene (TMS) was investigated using either bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system or using purified enzymes. TMS showed potent and selective inhibition of the ethoxyresorufin O-deethylation (EROD) activity of P450 1B1 with an IC(50) value of 6 nM. TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 (IC(50) = 300 nM) and 500-fold selectivity for P450 1B1 over P450 1A2 (IC(50) = 3 microM). The inhibitory effects of TMS on EROD activity of human liver microsomes were determined. TMS inhibited EROD activity of human liver microsomes at the same concentration as with recombinant human P450 1A2. TMS also strongly inhibited 4- and 2-hydroxylation of E(2) by P450 1B1-expressing membranes or purified P450 1B1. TMS was a competitive inhibitor of P450 1B1 with a K(i) of 3 nM. The inhibition by TMS was not mechanism-based, and the loss of activity was not blocked by the trapping agents glutathione, N-acetylcysteine, or dithiothreitol. Using purified histidine-tagged P450 1B1, the binding kinetic analysis was performed with TMS, yielding a K(d) of 3 microM. The activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline in an Escherichia coli lac-based mutagenicity tester system containing functional human P450 1B1 was strongly inhibited by TMS. Our results indicate that TMS is a very selective and potent competitive inhibitor of P450 1B1. TMS is selective for inhibiting P450 1B1 among other human P450s including 1A1, 1A2, and 3A4 and warrants consideration as a candidate for preventing mammary tumor formation by E(2) in humans.     

Dehydroepiandrosterone inhibits the expression of carcinogen-activating enzymes in vivo

We investigated the effect of the steroid hormone dehydroepiandrosterone (DHEA) on the hepatic expression and activity of carcinogen-activating enzymes, the cytochromes P450 (CYP) 1A1, 1A2 and 1B1, in Sprague-Dawley rats. In animals fed DHEA at 200 or 400 mg/kg body weight every other day for 2 weeks prior to exposure to the aryl hydrocarbon dimethylbenz[a]anthracene (DMBA, 5 mg/kg), there was a dose-dependent decrease in hepatic CYP activity, as measured by ethoxyresorufin-O (EROD) assay, from 37.1 to 22.9 and 14.7 pmoles/min/10 microg microsomes, respectively. DHEA did not directly inhibit microsomal EROD activity, however, leading us to investigate its effects on enzyme expression. To test this, we examined protein and mRNA levels of the enzymes. Western blot for CYP1A1 and CYP1A2 showed that DHEA inhibited the increase in hepatic CYP1A1 and CYP1A2 enzyme levels that are normally induced by DMBA. DMBA-induced increase in expression of CYP1A1, CYP1A2 and CYP1B1 mRNA was similarly blunted in DHEA-treated animals. DHEA was also able to significantly reduce the basal expression of CYP1A1 and CYP1A2 but not of CYP1B1. These results indicate that DHEA regulates the expression and, hence, the activity of hepatic carcinogen-activating enzymes in vivo, and this may be an important mechanism of its chemopreventive activity.     

Oral administration of naturally occurring coumarins leads to altered phase I and II enzyme activities and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of SENCAR mice

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.     

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice

Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y. Shimizu, Y. Nakatsuru, M. Ichinose, Y. Takahashi, H. Kume, J. Mimura, Y. Fujii-Kuriyama and T. Ishikawa (2000) PROC: Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice.     

Human lung microsomal cytochrome P4501A1 (CYP1A1) activities: impact of smoking status and CYP1A1, aryl hydrocarbon receptor, and glutathione S-transferase M1 genetic polymorphisms.

There are numerous conflicting epidemiological studies addressing correlations between cytochrome P450 1A1 (CYP1A1) genetic polymorphisms and lung cancer susceptibility, with associations plausibly linked to alterations in carcinogen bioactivation. Similarly, correlations between aryl hydrocarbon receptor gene (AHR) codon 554 genotype and CYP1A1 inducibility are controversial. The objective of this study was to determine whether smoking status, and CYP1A1, AHR, and glutathione S-transferase M1 gene (GSTM1) polymorphisms correlate with altered CYP1A1 activities. Lung microsomal CYP1A1-catalyzed 7-ethoxyresorufin O-dealkylation (EROD) activities were much higher in tissues from current smokers (n = 46) than in those from non-/former smokers (n = 24; 12.11 +/- 13.46 and 0.77 +/- 1.74 pmol/min/mg protein, respectively, mean +/- SD; P < 0.05). However, EROD activities in lung microsomes from current smokers CYP1A1*1/1 (n = 33) and heterozygous MspI variant CYP1A1*1/2A (n = 10) were not significantly different (12.23 +/- 13.48 and 8.23 +/- 9.76 pmol/min/mg protein, respectively, P > 0.05). Three current smokers were heterozygous variant CYP1A1*1/2B (possessing both *2A and *2C alleles), and exhibited activities similar to individuals CYP1A*1/1. One current smoker was heterozygous variant CYP1A1*4 and exhibited activities comparable with individuals CYP1A1*1/1 at that locus. EROD activities in microsomes from current smokers AHR(554)Arg/Arg (n = 41) and heterozygous variant AHR(554)Arg/Lys (n = 5) were not significantly different (12.13 +/- 13.56 and 12.01 +/- 14.23 pmol/min/mg protein, respectively; P > 0.05). Furthermore, microsomal EROD activities from current smokers with the GSTM1-null genotype (n = 28) were not significantly different from those (n = 18) carrying at least one copy of GSTM1 (12.61 +/- 14.24 and 11.34 +/- 12.53 pmol/min/mg protein, respectively; P > 0.05). Additionally, when genotypic combinations of CYP1A1, AHR, and GSTM1 were assessed, there were no significant effects on EROD activity. On the basis of microsomal enzyme activities from heterozygotes, CYP1A1*1/2A, CYP1A1*1/2B, CYP1A1*1/4, and AHR(554) Arg/Lys variants do not appear to significantly affect CYP1A1 activities in human lung, and we observed no association between CYP1A1 activity and the GSTM1-null polymorphism.     

Factors influencing elevation of intracellular Ca2+ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons.

Carcinogenic polycyclic aromatic hydrocarbons and a halogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were evaluated for their effects on intracellular Ca2+ in the human mammary epithelial cell line MCF-10A. After two 18-h incubations with MCF-10A cells, benzo[a]pyrene (BaP; 1, 3, and 10 microM) produced a dose-dependent increase in intracellular Ca2+. 7,12-Dimethylbenz[a]anthracene increased Ca2+ at 10 microM, whereas 3-methylcholanthrene and TCDD did not. The Ca2+-elevating effect of BaP appeared to be dependent on the influx of extracellular Ca2+, as addition of the Ca2+ chelator EGTA to the extracellular medium prevented the increase in Ca2+. MCF-10A cells were found by polymerase chain reaction to express cytochrome P4501A and P4501B isozymes as well as the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNAs associated with cytochrome P450 induction. Certain cytochrome P450-derived metabolites, including benzo[a]pyrene-7,8-diol (BP-diol) and benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), were more effective in increasing Ca2+ than was BaP. The Ca2+-elevating effect of BP-diol was prevented by alpha-naphthoflavone, a cytochrome P4501A and P4501B inhibitor, but not by the antioxidant N-acetylcysteine. These results suggest that cytochrome P450-dependent formation of BPDE from BP-diol is a major mechanism required for elevation of Ca2+ in MCF-10A cells.     

Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats.

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, has been shown to inhibit chemical carcinogenesis, possibly due to its ability to block the activation or to enhance the detoxification of chemical carcinogens. The present study was conducted to elucidate the biochemical mechanisms involved by characterizing the effects of PEITC on phase I and phase II xenobiotic-metabolizing enzymes. A single dose of PEITC to F344 rats (1 mmol/kg) decreased the liver N-nitrosodimethylamine demethylase (NDMAd) activity (mainly due to P450 2E1) by 80% at 2 h and the activity of NDMAd remained decreased by 40% at 48 h after treatment. The liver pentoxyresorufin O-dealkylase (PROD) activity and P450 2B1 protein level were elevated 10- and 7-fold at 24 h after treatment respectively. The liver microsomal ethoxyresorufin O-dealkylase (EROD) (mainly due to P450 1A) and erythromycin N-demethylase (mainly due to P450 3A) activities were decreased at 2-12 h after treatment and recovered afterwards. The lung microsomal PROD and EROD activities were not significantly affected; whereas, the nasal microsomal PROD and EROD activities were decreased by 40-50%. After a treatment with PEITC, the rates of oxidative metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were decreased in liver microsomes by 40-60% at 2 h and recovered gradually; the rates in lung microsomes were markedly decreased by 60-70% at 2 h and remained at the decreased level at 24 h; and the rates in nasal mucosa microsomes were decreased gradually with the lowest activities observed at 18 h (50%) followed by a gradual recovery. Furthermore, the treatment with PEITC resulted in a maximal 5-fold increase of NAD(P)H:quinone oxidoreductase and 1.5-fold increase of glutathione S-transferase activities in the liver, but the activities of these two enzymes were not significantly affected in the lung and nasal mucosa. The sulfotransferase activity in the liver was decreased by 32-48% at 24-48 h after treatment; the nasal activity was increased by 1.8- to 2.5-fold, but the lung activity was not significantly changed. The hepatic UDP glucuronosyltransferase activity was slightly decreased at 2 h but slightly increased at 48 h after treatment, but no changes were observed for the lung and nasal activities. The study demonstrates that PEITC selectively affects xenobiotic-metabolizing enzymes in the liver, lung and nasal mucosa and it is especially effective in inhibiting the P450-dependent oxidation of NNK in the lung and of NDMA in the liver.     

Characterization of a novel cytochrome P450 from the transformable cell line, C3H/10T1/2

The cytochrome P450 in the transformable C3H/10T1/2 (10T1/2) cell line has been characterized and compared to the major polycyclic aromatic hydrocarbon (PAH)-inducible hepatic form, cytochrome P450IA1 (P450IA1). The mouse hepatoma cell line, Hepa-1, was used as an in vitro model for P450IA1 expression and regulation by PAH. Microsomes from uninduced and benz[a]anthracene (BA)-induced 10T1/2 cells provided PAH mono-oxygenated product profiles that were totally different from metabolite profiles produced by microsomes from uninduced and BA-induced Hepa-1 cells even though total activities were similar. The proximate carcinogen, 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) was a major product for the 10T1/2 microsomes, while Hepa-1 formed less than 2% of this metabolite. Hepa-1 converted benzo[a]pyrene (BP) to BP-4,5-diol and DMBA to 7-hydroxymethyl-12-methyl-BA, while 10T1/2 did not produce either product. Polyclonal antibody to rat hepatic P450IA1 did not inhibit metabolism of either PAH substrate by 10T1/2 microsomes, but totally inhibited such metabolism by Hepa-1 microsomes. Western immunoblot analysis of BA-induced 10T1/2 microsomes showed that less than 1% of total P450 was P450IA1. The PAH-metabolizing activity of 10T1/2 microsomes was highly inducible (14-fold) by pre-treatment of non-confluent intact cells with BA, but was only half as inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, the P450IA1 activity of Hepa-1 cells was highly inducible by both compounds. The distinct metabolite profiles, antibody inhibition data and lack of immunoreactivity all indicate that PAH metabolism in 10T1/2 cells is catalyzed by a form of P450 distinct from P450IA1. The anomalous induction patterns suggest that this novel isozyme is predominantly regulated by a mechanism other than the Ah receptor.