Elovaara Eivor, Väänänen Virpi, Mikkola Jouni
Laboratory of Toxicokinetics and Metabolism, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, 00250, Helsinki, Finland.
Arch Toxicol. 2003 Apr;77(4):183-93. doi: 10.1007/s00204-003-0436-0. Epub 2003 Feb 12.
Two fluorimetric HPLC methods are described for the quantification of naphthols, phenanthrols and 1-hydroxypyrene (1-OHP) in urine specimens obtained from male Wistar rats exposed to naphthalene, phenanthrene and pyrene. The polycyclic aromatic hydrocarbons (PAHs) were given intraperitoneally, either alone (1.0 mmol/kg body weight) or as an equimolar mixture (0.33 mmol/kg), using the same dosages for repeated treatments on week 1 and week 2. Between these treatments, PAH-metabolizing activities encoded by aryl hydrocarbon (Ah) receptor-controlled genes were induced in the rats with beta-naphthoflavone (betaNF). Chromatographic separation of five phenanthrols (1-, 2-, 3-, 4-, and 9-isomers) was accomplished using two different RP C-18 columns. Despite selective detection (programmable wavelengths), the quantification limits in the urine ranged widely: 1-OHP (0.18 microg/l) <phenanthrols (0.34-0.45 microg/l) <2-naphthol (1.5 microg/l) <1-naphthol (4 micro g/l). The relative standard deviation of the methods was good, as also was the reproducibility. The molar fraction of the dose excreted in 24-h urine as naphthols (<or=4.0%), phenanthrols (<or=1.1%), and 1-OHP (<or=2.4%) was low. Urinary disposition increased differentially in betaNF-induced rats: naphthols, 9-phenanthrol (1- to-2-fold); 2-, 3-, and 4-phenanthrols (4- to 5-fold); 1-phenanthrol and 1-OHP (over 11-fold). The OH-metabolites were analyzed before and after enzymatic hydrolysis (beta-glucuronidase/arylsulfatase). The percentage excreted as a free phenol in urine varied for 1-OHP (2-11%), 1-naphthol (36-51%), 2-naphthol (59-65%), and the phenanthrols (29-94%). 1-Naphthyl- and 1-pyrenyl beta- d-glucuronide served as measures for the completeness of enzymatic hydrolysis. Characteristic differences observed in the urinary disposition of naphthalene, phenanthrene, and pyrene are described, as well as important factors (dose, metabolic capacity, relative urinary output) associated with biomarker validation. This intervention study clarifies intraindividual variation in PAH metabolism and provides useful information for the development of new methods applicable in the biomonitoring of PAH exposure in humans.
本文描述了两种荧光高效液相色谱法，用于定量分析从接触萘、菲和芘的雄性Wistar大鼠获取的尿液样本中的萘酚、菲酚和1-羟基芘（1-OHP）。多环芳烃（PAHs）通过腹腔注射给药，单独给药（1.0 mmol/kg体重）或作为等摩尔混合物（0.33 mmol/kg），在第1周和第2周使用相同剂量进行重复处理。在这些处理之间，用β-萘黄酮（βNF）诱导大鼠体内由芳烃（Ah）受体控制的基因编码的PAH代谢活性。使用两种不同的反相C-18柱完成了五种菲酚（1-、2-、3-、4-和9-异构体）的色谱分离。尽管采用了选择性检测（可编程波长），但尿液中的定量限差异很大：1-OHP（0.18 μg/l）＜菲酚（0.34 - 0.45 μg/l）＜2-萘酚（1.5 μg/l）＜1-萘酚（4 μg/l）。方法的相对标准偏差良好，重现性也很好。以萘酚（≤4.0%）、菲酚（≤1.1%）和1-OHP（≤2.4%）形式排泄到24小时尿液中的剂量摩尔分数较低。在βNF诱导的大鼠中，尿液处置情况有差异地增加：萘酚、9-菲酚（增加1至2倍）；2-、3-和4-菲酚（增加4至5倍）；1-菲酚和1-OHP（增加超过11倍）。在酶促水解（β-葡萄糖醛酸酶/芳基硫酸酯酶）前后对OH-代谢物进行了分析。以游离酚形式排泄到尿液中的百分比在1-OHP（2% - 11%）、1-萘酚（36% - 51%）、2-萘酚（59% - 65%）和菲酚（29% - 94%）中各不相同。1-萘基-β-D-葡萄糖醛酸苷和1-芘基-β-D-葡萄糖醛酸苷用作酶促水解完整性的指标。描述了在萘、菲和芘的尿液处置中观察到的特征差异，以及与生物标志物验证相关的重要因素（剂量、代谢能力、相对尿量）。这项干预研究阐明了PAH代谢的个体内差异，并为开发适用于人体PAH暴露生物监测的新方法提供了有用信息。
