高效液相色谱法测定外周血BPDE-DNA加合物的含量

【目的】建立高效液相色谱（high performance liquid chromatographv,HPLC）紫外检测法测定反式二羟基环氧苯并（a）芘[（＋）-anti-benzoa）pyrene diol-epexide,BPDE]在人体内形成的BPDE—DNA加合物的含量,对其质量进行控制。【方法】采用Kromasil—C18柱（150mm×4．6mm,5μm）为分析柱,以甲醇-水-磷酸（80：20：0．1,v／v）为流动相,流速1．0ml／min紫外检测器检测,检测波长254nm。【结果】在4．016-803．2ng／ml的浓度范围内,BPDE峰面积与浓度呈良好的线性,相关系数r=0．9997;平均回收率均在85％～115％之间,RSD为1．32％-1．72％。【结论】本法准确、方便,可用于测定空气中苯并（a）芘在体内代谢产物BPDE～DNA加合物的含量。     

Tissue Distribution and Metabolism of Benzo[a]pyrene in Embryonic and Larval Medaka (Oryzias latipes)

The need to understand chemical uptake, distribution, and metabolismin embryonic and larval fish derives from the fact that theseearly life stages often exhibit greater sensitivity to xenobioticcompounds than do adult animals. In this study, a 6-h acutewaterborne exposure immediately after fertilization was usedto quickly load the egg with benzo[a]pyrene (BaP). This exposurewas used to mimic the initial egg concentration of a persistentbioaccumulative toxicant that could result from maternal transfer.We used multiphoton laser scanning microscopy (MPLSM) in combinationwith conventional analytical chemistry methods to characterizethe tissue distribution of BaP and its principal metabolitesin medaka embryos and post-hatch larvae. Embryonic metabolismof BaP was evident by MPLSM prior to liver formation or heartdevelopment. A major product of this metabolism was identifiedby liquid chromatography/mass spectrometry as BaP-3-glucuronide.MPLSM showed that metabolites were sequestered within the yolk,biliary system, and gastrointestinal tract. When the gastrointestinaltract became patent a few days after hatch, the metaboliteswere rapidly eliminated. These findings indicate that some ofthe earliest embryonic tissues are metabolically competent andthat redistribution of BaP and its metabolic products occursthroughout development. Rapid metabolism of BaP substantiallyreduces the body burden of parent chemical in the developingembryo, potentially reducing toxicity. It remains unclear whethermetabolism of BaP in medaka embryos leads to the formation ofDNA adducts associated with genotoxic effects or yields metabolitesthat later lead to other toxicity in juveniles or adults.     

苯并（a）芘对肝细胞凋亡及脂质过氧化作用

目的探讨苯并（a）芘（BaP）对小鼠肝细胞凋亡及其与脂质过氧化的关系。方法ICR小鼠灌胃染毒,染毒剂量分别为0、5、10、20,40mg／kg,应用吖啶橙／溴化乙啶（AO／EB）荧光双染法检测肝细胞凋亡情况并测定肝组织脂质过氧化主要终产物丙二醛（MDA）的含量。结果BaP各染毒剂量组肝细胞凋亡发生率与对照组比较,差异均有统计学意57．（P〈0.05）,MDA含量40mg／kg BaP染毒剂量组与对照组比,较差异具有统计学意义（P〈0.05）。结论BaP可诱发小鼠肝细胞发生凋亡和脂质过氧化,氧化损伤参与了介导细胞凋亡的过程。     

Coordinate regulation of human drug-metabolizing enzymes, and conjugate transporters by the Ah receptor, pregnane X receptor and constitutive androstane receptor

Coordinate regulation of Phase I and II drug-metabolizing enzymes and conjugate transporters by nuclear receptors suggests that these proteins evolved to an integrated biotransformation system. Two major groups of ligand-activated nuclear receptors/xenosensors evolved: the Ah receptor (activated by aryl hydrocarbons and drugs such as omeprazole) and type 2 steroid receptors such as PXR and CAR, activated by drugs such as rifampicin, carbamazepin and phenytoin. It is increasingly recognized that there is considerable cross-talk between these xenosensors. Therefore, an attempt was made to discuss biotransformation by the Ah receptor together with that of PXR and CAR. Due to considerable species differences the emphasis is on human biotransformation. Agonists coordinately induce biotransformation due to common xenosensor-binding response elements in the regulatory region of target genes. However, whereas different groups of xenobiotics appear to more selectively stimulate CYPs (Phase I), their regulatory control largely converged in modulating Phase II metabolism and transport. Biotransformation appears to be tightly controlled to achieve efficient homeostasis of endobiotics and detoxification of dietary phytochemicals, but nuclear receptor agonists may also lead to potentially harmful drug interactions.     

Benzopyrene exposure disrupts DNA methylation and growth dynamics in breast cancer cells

Exposures to environmental carcinogens and unhealthy lifestyle choices increase the incidence of breast cancer. One such compound, benzo(a)pyrene (BaP), leads to covalent DNA modifications and the deregulation of gene expression. To date, these mechanisms of BaP-induced carcinogenesis are poorly understood, particularly in the case of breast cancer. We tested the effects of BaP exposure on cellular growth dynamics and DNA methylation in four breast cancer cell lines since disruptions in DNA methylation lead to deregulated gene expression and the loss of genomic integrity. We observed robust time- and concentration-dependent loss of proliferation, S phase and G2M accumulation and apoptosis in p53 positive MCF-7 and T47-D cells. We observed minimal responses in p53 negative HCC-1086 and MDA MB 231 cells. Furthermore, BaP increased p53 levels in both p53 positive cell lines, as well as p21 levels in MCF-7 cells, an effect that was prevented by the p53-specific inhibitor pifithrin-alpha. No changes in global levels of DNA methylation levels induced by BaP were detected by the methyl acceptor assay (MAA) in any cell line, however, methylation profiling by AIMS (amplification of intermethylated sites) analysis showed dynamic, sequence-specific hypo- and hypermethylation events in all cell lines. We also identified BaP-induced hypomethylation events at a number of genomic repeats. Our data confirm the p53-specific disruption of the cell cycle as well as the disruption of DNA methylation as a consequence of BaP treatment, thus reinforcing the link between environmental exposures, DNA methylation and breast cancer.     

Effect of a flue-curing process that reduces tobacco specific nitrosamines on the tumor promotion in SENCAR mice by cigarette smoke condensate.

A 30-week dermal tumor promotion study was conducted to evaluate the dermal tumor-promoting potential of cigarette smoke condensate (CSC) collected from cigarettes containing flue-cured tobacco cured by a heat-exchange process (HE) relative to that of cigarettes containing flue-cured tobacco cured by the traditional direct-fire process (DF). Heat-exchange process cured tobacco contains significantly lower concentrations of tobacco specific nitrosamines (TSNAs) compared to traditional direct-fire cured tobacco. Mainstream CSCs were collected by cold trap from smoke generators using the Federal Trade Commission puffing regimen. Groups of 40 female SENCAR mice were initiated by a single application of 75 micro g 7,12-dimethylbenz[a]anthracene (DMBA) to the shaved dorsal skin. CSCs were then applied to the skin three times/week for 29 weeks at 9, 18, or 36mg tar/application. End-points included body weights, clinical observations, organ weights, dermal tumor development and histopathology. The numbers of dermal tumors and the numbers of tumor-bearing mice for each CSC were statistically different from the DMBA/acetone control group and increased with increasing dose. When corresponding doses of each CSC were compared, only the DMBA/mid-dose HE CSC group was statistically significantly different (lower) from the corresponding DMBA/mid-dose DF CSC group. In this assay, the dermal tumor-promotion potential of CSC from heat-exchange flue-cured tobacco did not differ from that of traditional direct-fire flue-cured tobacco CSC.     

Concentration dependent effects of tobacco particulates from different types of cigarettes on expression of drug metabolizing proteins, and benzo(a)pyrene metabolism in primary normal human oral epithelial cells

The ability of tobacco smoke (TS) to modulate phase I and II enzymes and affect metabolism of tobacco carcinogens is likely an important factor in its carcinogenicity. For the first time several types of TS particulates (TSP) were compared in different primary cultured human oral epithelial cells (NOE) for their abilities to affect metabolism of the tobacco carcinogen, (BaP) to genotoxic products, and expression of drug metabolizing enzymes. TSP from, reference filtered (2RF4), mentholated (MS), reference unfiltered, (IR3), ultra low tar (UL), and cigarettes that primarily heat tobacco (ECL) were tested. Cells pretreated with TSP concentrations of 0.2-10 μg/ml generally showed increased rates of BaP metabolism; those treated with TSP concentrations above 10 μg/ml showed decreased rates. Effects of TSPs were similar when expressed on a weight basis. Weights of TSP/cigarette varied in the order: MS ≈ IR3 > 2RF4 > ECL > UL. All TSPs induced the phase I proteins, cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), phase II proteins, NAD(P)H dehydrogenase quinone 1 (NQO1), and microsomal glutathione S-transferase 1 (MGST1), and additionally, hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2), as assessed by qRT-PCR. The pattern of gene induction at probable physiological levels favored activation over detoxification.     

Characterization of calcium oscillations in normal and benzo[a]pyrene-treated clone 9 cells.

Intracellular Ca2+ oscillations induced by oxytocin and vasopressin were analyzed in a rat liver cell line (Clone 9) in order to identify mechanisms by which benzo[a]pyrene (BaP) alters Ca2+ signaling patterns in these cells. Clone 9 cells exhibit an initial Ca2+ spike, followed by Ca2+ oscillations upon oxytocin or vasopressin treatment. The range of frequencies (maximum 110 mHz) was dependent on agonist concentration with a constant amplitude less than or equal to the amount of Ca2+ generated from the inositol trisphosphate (InsP(3))-sensitive pool. This study examined contributions of extracellular and intracellular pools to the frequency of Ca2+ oscillations and the role of membrane channels, second messengers, and different pharmacological reagents on the regulation of oscillation frequency in both control and BaP-treated cells. Results indicated that the Ca2+ oscillations are mainly due to inositol 1,4,5-triphosphate (InsP(3))-sensitive stores and that extracellular Ca2+ contributes to refilling of this intracellular Ca2+ pool. The frequency of Ca2+ oscillations is also sharply affected by protein kinase C activated by phospholipase C. In BaP-treated Clone 9 cells, basal Ca2+ levels were elevated and the frequency of Ca2+ oscillations was suppressed in a dose-dependent fashion. Suppression of Ca2+ oscillations is due, at least in part, to an effect of BaP on enhanced opening of K+ channels. This was confirmed by showing that inhibition of the K+ channel opening by tetraethylammonium chloride can reverse the effect of BaP on oxytocin-induced Ca2+ oscillations, and potentially decrease the toxicity of BaP.     

Effects of dietary broccoli and butylated hydroxyanisole on liver-mediated metabolism of benzo[a]pyrene

Groups of male Wistar rats were given either a basal diet or diets supplemented with 10 or 25% broccoli or 0.8% BHA. Liver fractions were assayed for cytochrome P-450, for aryl hydrocarbon hydroxylase (AHH), glutathione-S-transferase and epoxide hydrolase activities and for benzo[a]pyrene (BaP) metabolism. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of liver microsomes was also performed. Mean relative liver weight in the BHA group was significantly higher than that of the control and 10% broccoli groups but not significantly higher than that of the 25% broccoli group. Gel electrophoresis of liver microsomes indicated a diet-dependent variation in intensity in a band that corresponded in mol wt to those of certain cytochrome P-450s. Diet-dependent increases (20-90% over control) in cytochrome P-450 and in the activities of AHH, glutathione-S-transferase and epoxide hydrolase were observed in livers from rats given broccoli-supplemented diets. Except for AHH activity, such increases also occurred in the group fed BHA. Analysis of BaP metabolites revealed that the proportion of 4,5-diol formed relative to the major diols identified was unchanged in the broccoli- or BHA-treated groups relative to the control group. The proportion of 9,10-diol formed was unchanged in the broccoli-fed groups but was significantly higher in the BHA group than in the control group. The proportion of cis and trans-7,8-diol formed was unchanged in both broccoli-fed groups but was significantly lower in the BHA group. In comparison with the control group, the ratio of phenol I (comprising primarily 9-OH-BaP) to total phenols (primarily 9-OH and 3-OH) was significantly decreased by about 30% in the 25%-broccoli group and by about 70% in the BHA group. Qualitative differences in the phenol-II peak (comprising 3-OH and 7-OH phenols) were also observed between samples from the controls and those of 25%-broccoli- and BHA-fed rats. The implications of these findings are discussed with respect to the effects of broccoli and BHA on benzo[a]pyrene toxicity.