Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method

A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP

Chemical Methods for Determination of Hydroxylated Metabolites of Polycyclic Aromatic Hydrocarbons and Polychlorinated Biphenyls in Biological Material

U ovome preglednom radu prikazani su postupci analize hidroksiliranih metabolita policikličkih aromatskih ugljikovodika i poliklorbifenila u ljudima i životinjama. Ti metaboliti služe kao biomarkeri izloženosti ljudi i životinja navedenim zagađivalima, no neki od njih i sami posjeduju toksična svojstva. Analiziraju se najčešće u urinu koji je kao uzorak najdostupniji, ali se isto tako mogu analizirati i u ljudskoj, odnosno životinjskoj jetri, žuči i masnom tkivu. Analiza metabolita aromatskih zagađivala važna je zbog određivanja biodostupnosti aromatskih zagađivala, njihove potencijalne toksičnosti u ljudskom organizmu, ali i zbog toksičnosti samih metabolita. Napredak analitičkih metoda omogućio je simultanu analizu velikog broja metabolita u uzorcima. Nove tehnike ekstrakcije i selektivnije i preciznije kvalitativne i kvantitativne analize omogućuju detekciju vrlo niskih koncentracija metabolita. Pri tome dodatnu prednost imaju jednostavne tehnike koje zahtijevaju manje kemikalija i vremena za analizu.This review presents methods for the analysis of hydroxylated metabolites of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in humans and animals. These metabolites serve as biomarkers of human and animal exposure to the mentioned pollutants, but some metabolites also have toxic properties. Most are analysed in urine, which is the most accessible sample, but they can also be analysed in human and animal liver, bile, and adipose tissue. Their analysis is important for assessing bioavailability of aromatic pollutants and their toxicity in human organism, but also the toxicity of metabolites themselves. Advancements in analytical methods have made it possible to analyse multiple metabolites in a sample at the same time. New extraction techniques and more precise and selective qualitative and quantitative analyses can now detect very low metabolite oncentrations. An extra advantage is that these simple techniques require less chemicals and time

Common and distinct mechanisms underlying the establishment of adaxial and abaxial polarity in stamen and leaf development

Establishment of adaxial-abaxial polarity is essential for lateral organ development. A stamen consists of a bilaterally symmetrical anther and a radial filament. Using a rice mutant, rod-like lemma, in which establishment of adaxial-abaxial polarity is compromised, we found that stamen patterning is likely to be achieved by a unique regulatory mechanism: rearrangement of adaxial-abaxial polarity in the anther, and abaxialization in the filament. These regulations are not found in leaf development. Here, we discuss similarities and differences between the stamen and the leaf in the mechanisms underlying the establishment of adaxialabaxial polarity. In addition, we propose the idea that the process of establishing adaxial-abaxial polarity in lateral organs is likely to be divided into two phases: a meristem-dependent, followed by a meristem-independent phase. In stamen development, the transition between these two phases is clearly observed as the rearrangement of expression patterns of the adaxial and abaxial marker genes