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Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.

The production of labeled brominated metabolites with radioactive 82 Br in Laurencia species was investigated as part of a study of the biosynthesis of halogenated metabolites from species belonging to the red algal genus Laurencia (Rhodomelaceae, Ceramiales Br-containing metabolites. In the case of the laurencin-producing race of L. nipponica, laurencin and deacetyllaurencin were found to be produced in approximately 1:1 ratio, though laurencin is the major metabolite in the wild sample. Furthermore, when cultured in the dark, the production rates of brominated metabolites in Laurencia spp. were found to be diminished. The present study strongly indicates that the use of radiobromine [ 82 Br] in combination with the TLC-ARLG method is an effective approach for investigating the biosynthesis of brominated metabolites in Laurencia.3

The present method involving an acid-base extraction, in situ derivatization, and GC-MS analysis has shown to be a simple, efficient, and reliable method for the isolation and identification of the chemical substances in DEP.

Goal, Scope, and Background We recently developed a new isolation method for diesel exhaust particles (DEP), involving successive extraction with H 2 O, sodium bicarbonate, and sodium hydroxide, in which the sodium hydroxide extract was found to consist of phenolic components. Analysis of the extract revealed that vasodilative active nitrophenols are in DEP in significantly higher concentration than those estimated by an earlier method involving a combination of solvent extraction and repeated chromatography. These findings indicated that our new procedure offers a simple, efficient and reliable method for the isolation and identification of bioactive substances in DEP. This encouraged us to extend our work to investigating new vasodilatory substances in the sodium bicarbonate extract. Materials and Methods DEP were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). GC-MS analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan).Results DEP dissolved in 1-butanol was successively extracted with water, sodium bicarbonate, and then aqueous sodium hydroxide. The sodium bicarbonate extract was neutralized and the resulting mixture of acidic components was subjected to reverse-phase (RP) column chromatography followed by RP-HPLC with fractions assayed for vasodilative activity. This led to the identification of telephthalic acid, p-hydroxybenzoic acid, isophthalic acid, phthalic acid, 3-hydroxy-4-nitrobenzoic acid, 4-hydroxy-3-nitrophenol, and 1,4,5-naphthalene tricarboxylic acid as components of DEP. DiscussionThe sodium bicarbonate extract was rich in the acidic components. Repeated reverse-phase chromatography resulted in the successful isolation of several acidic substances including the new vasodilative materials, 4-hydroxy-3-nitrobenzoic acid and 3-hydroxy-4-nitrobenzoic acid. Seidel et al. 2006). A number of studies on the adverse effects of diesel exhaust particles (DEP) on human health, including lung cancer (McClellan 1987;Ichinose et al. 1997), allergic rhinitis (Muranaka 1986;Takafuji et al. 1987), bronchial asthma-like disease (Sagai et al. 1996;Miyabara et al. 1998), and disruption of endocrine function, have been reported (Yoshida et al. 1999;Watanabe & Oonuki 1999;Tsukue et al. 2001Tsukue et al. , 2002. While these studies have furnished epidemiologically important results, they have been done without knowledge of the bioactive entities in DEP, making it difficult to estimate the effect of individual components on human health. In our earlier studies on the isolation and identification of bioactive compounds contained in DEP, we have isolated aromatic substances including nitrophenols (Mori et al. 2003a;Taneda et al. 2004a) that showed strong vasodilatory activity by a method involving successive solvent extractions, followed by repeated column chromatography. This multi-step isolation procedure, however, resulted in poor fractionation with impaired accuracy and reproducibility. Therefore, it was crucial to develop a more efficient and ...