NITROGEN HETEROCYCLES: METABOLIC EFFECTS AND TOXICITY

氮杂环：代谢效应和毒性

• 批准号: 3251211
• 负责人: RAYMOND F NOVAK
• 金额: $ 22.68万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3251211
• 关键词: SDS polyacrylamide gel electrophoresis; actins; autoradiography; carbon; chemical carcinogenesis; coffee; complementary DNA; cytochrome P450; drug adverse effect; genetic translation; goats; heterocyclic compounds; immunocytochemistry; kidney; laboratory mouse; laboratory rabbit; laboratory rat; messenger RNA; microsomes; necrosis; nitrogen compounds; nitrogen metabolism; nitrogen oxides; occupational hazard; oligonucleotides; oxygenases; peroxisome; pesticides; protein biosynthesis; pyridine; radionuclides; radiotracer; ribosomes; sulfur; toxin metabolism; tritium; tubulin; western blottings

Pyridine (PY) is present in coal tar, tobacco smoke, roasted coffee and creosote oil. PY inhalation has been reported to increase liver:body weight ratios in rats and to produce fatty degeneration of the liver; oral administration of PY to rats caused hepatic necrosis and the appearance of hepatic nodules. Research in our laboratories has shown PY to be a potent inducer of cytochromes P-450 in nasal, hepatic and renal tissues following inhalation exposure or i.p. administration. Western blot analysis shows P450IIE1 to be one of the major forms induced in these tissues and this form is the low KM PY N-oxidase. Preliminary data reveal no increase in hybridization P450IIE1 mRNA levels in total RNA isolated from hepatic tissues as evidence by Northern or dot blot analysis following PY treatment. In contrast, Northern or dot blot analysis reveals a rapid time-dependent loss of P450IIE1 poly (A)+ mRNA in hepatic tissue following PY treatment. This decrease in poly(A)+ mRNA is attributed to a loss of the poly(A)+ tail and is associated with early posttranscriptional events (increased translational efficiency) of P450IIE1 induction. Moreover, immunocytochemistry shows the presence of P450IIE1 in the nasal "transition zone" and initial data suggest that PY may be per- oxisome proliferator. Thus, research is proposed to examine two hypotheses: (1) that P450IIE1 exhibits a unique substrate specificity in the oxidation of certain N and S atoms in xenobiotics, and (2) that PY induction of P450IIE1 occurs by posttranscriptional events, i.e. by increased translational efficiency which results in increased protein synthesis, whereas simultaneous induction of P450IIE1 and IA2 by PY is mediated by both transcriptional activation and posttranscriptional events. The specific objectives include: studies on the substrate specificity of P450IIE1 toward N- and S- containing xenobiotics, immunohistochemical studies on PY induction of P450 in nasal tissue including examination of P450IIE1, IA1 and IA2 mRNA in situ; and examination of the role of transcriptional and posttranscriptional regulation of P450IIE1, IA1 and IA2 in nasal, hepatic and renal tissue. Northern and dot blot analysis of mRNA and poly(A)+ mRNA, nuclear run-off assays, cell free translation of poly(A)+ mRNA, analysis of poly(A)+ tail length, mRNA ribosomal content, the levels of polysome-bound and free mRNA and the rate of P-450 protein synthesis in the presence and absence of cyclohexamide and actinomycin D following PY treatment will be accomplished with the goal of elucidating posttranscriptional regulatory pathways in the induction of P450s by PY.

吡啶（PY）存在于煤焦油、烟草烟雾、烘焙咖啡和 杂酚油 据报道，吸入PY可增加肝脏：身体 大鼠体重比并产生肝脏脂肪变性;经口给药 PY给药大鼠引起肝坏死， 肝结节 我们实验室的研究表明，PY是一种有效的 鼻、肝和肾组织中细胞色素P-450的诱导剂 吸入暴露或i. p.给药。 蛋白质印迹分析显示， P450 IIE 1是这些组织中诱导的主要形式之一， 形式是低KM PY N-氧化酶。 初步数据显示， 肝组织总RNA中P450 IIE 1 mRNA水平的杂交 组织作为PY后北方或斑点印迹分析的证据 治疗 相比之下，北方或斑点印迹分析揭示了快速的 肝组织中P450 IIE 1 poly（A）+ mRNA的时间依赖性丢失 PY治疗。 poly（A）+ mRNA的这种减少归因于 poly（A）+尾，与早期转录后事件相关 （增加的翻译效率）的P450 IIE 1诱导。 此外，委员会认为， 免疫细胞化学显示P450 IIE 1在鼻“过渡区”中的存在 区域”和初步数据表明PY可能是过氧化物酶体增殖剂。 因此，本研究提出了两个假设：（1）P450 IIE 1 在某些N和S的氧化中表现出独特的底物特异性 异生物质中的原子，和（2）PY诱导P450 IIE 1发生， 转录后事件，即通过提高翻译效率 这导致蛋白质合成增加，而同时 PY对P450 IIE 1和IA 2诱导作用是由两种转录因子介导的， 激活和转录后事件。 的具体目标 包括：研究P450 IIE 1对N-和S-的底物特异性 免疫组化研究PY对P450的诱导作用 鼻组织中P450 IIE 1、IA 1和IA 2 mRNA的表达， 原位;和检查转录和 P450 IIE 1、IA 1和IA 2在鼻、肝、 和肾组织。 mRNA和poly（A）+的北方和斑点印迹分析 mRNA、核径流测定、poly（A）+ mRNA的细胞游离翻译， 分析poly（A）+尾长、mRNA核糖体含量、 多核糖体结合和游离mRNA和P-450蛋白合成率在 PY后是否存在环己脲和放线菌素D 治疗的目的是阐明 PY诱导P450的转录后调节途径。