Bioassay-directed fractionation

Nishioka MG, CC Howard, DA Conros, LM Ball (1988) Detection of hydroxylated nitro aromatic and hydroxy-lated nitro polycyclic aromatic compounds in ambient air particulate extract using bioassay-directed fractionation. Environ Sci Technol 22 908-915. 

Fig. 3.18. Scheme for the bioassay-directed fractionation/chemical analysis performed with the sludge extract. Reprinted with permission from G. A. Umbuzeiro et al. [97]. 

One especially successful method of testing complex mixtures is bioassay-directed fractionation followed by chemical identification of active compounds. Until now this method has mainly been used for the testing and identification of genotoxic compounds in environmental mixtures such as extracts of air particulates, exhaust condensates, and cooked foods. In this approach, each fraction is bioassayed untd the major class of specihc chemical(s) responsible for the activity can be isolated and chemically characterized, which make a risk assessment of the mixture possible. 

Throughout this chapter, we cite examples of the use of the NIST Standard Reference Material SRM 1649, which is referred to as Air Particles or Urban Air Particulate Matter, (a) to validate analytical procedures for determination of PAHs and PACs in samples of complex mixtures of particulate matter in ambient air and (b) for laboratory intercomparisons of methodologies for bacterial bioassays and bioassay-directed fractionations of organic extracts of such mixtures (e.g., see Claxton et al., 1992a Lewtas et al., 1990a, 1992 and May et al., 1992). 

Durant and co-workers (1998) analyzed an organic extract of the NIST reference complex mixture SRM 1649 (see Box 10.3) using the same human cell line (hlAlv2) and bioassay-directed fractionation. In the nonpolar fraction, cy c 1 o pe n t a[ cz/ ] py re n e, benzo[a]-pyrene, and benzo[ b ]fl uo ran the ne were responsible for 7, 4, and 2%, respectively, of the total extract mutagenicity. Only one potent O-PAC mutagen was identified in the semipolar fraction, the ketone, 6H-benzo[cd ]pyren-6-one ( 0.5%) (see discussion in Section D.3). 

Figure 10.25 shows these mutagrams for the vapor and particle phases, respectively. Interestingly, the total direct mutagenicity of the vapor phase, 210 rev m-3, was actually greater than that of the particle phase, 160 rev m 3 furthermore, its mutagenicity profile was substantially different. Thus, fraction 4 is the major peak for the vapor-phase sample whereas most of the particle-phase mutagenicity is in the more polar peaks 6 and 7. Similar enhancements in the contributions of more polar species were reported for bioassay-directed fractionation of SRM 1649 urban air particulate matter (Schuetzle and Lewtas, 1986 Nishioka et al., 1988 ... 

Bioassay-directed fractionations of the daytime and nighttime samples produced the mutagrams shown in Fig. 10.26. The ambient nitronaphthalenes and methylnitronaphthalenes in fraction 4 contribute 18% of the total daytime mutagenic activity of 23 rev m-3 they are formed by OH radical attack on the parent PAHs (see Sasaki et al., 1995, and Section F). The total nighttime activity is higher than the daytime activity, 31 rev m-3, and was attributed to more efficient formation of the nitronaphthalenes and methylnitronaphthalenes in NO, radical initiated reactions (Atkinson and Arey, 1994). ... 

For example, Legzdins and co-workers (1994) used the bioassay-directed fractionation and chemical analysis technique to isolate, identify, and quantify 2-nitrofluoranthene in extracts of ambient particles collected in Hamilton, Ontario, Canada. They found it accounted for 70% of the total nonpolar direct bacterial mutagenicity (strain YG1021, standard reversion assay, Maron and Ames, 1983). 

Arey, J., W. P. Harger, D. Helmig, and R. Atkinson, Bioassay-Directed Fractionation of Mutagenic PAH Atmospheric Photooxidation Products and Ambient Particulate Extracts, Mutat. Res., 281, 67-76 (1992). 

Chuang, J., M. Nishioka, and B. Petersen, Bioassay-Directed Fractionation of the Organic Extract of SRM 1649 Urban Air Particulate Matter, Int. J. Environ. Anal. Chem., 39, 245-256 (1990). 

J. Butler, and J. Louis, Bioassay-Directed Fractionation of Organic Compounds Associated with Airborne Particulate Matter An Interseasonal Study, Atmos. Environ., 27A, 1609-1626(1993). 

Lewtas, J., L. King, K. Williams, L. Ball, and D. DeMarini, Bioassay-Directed Fractionation of 1-Nitropyrene Metabolites Generation of Mutagrams by Coupling Reverse-Phase HPLC with Microsuspension Mutagenicity Assays, Mutagenesis, 6, 481-489 (1990b). 

Nishioka, M. G., C. C. Howard, D. A. Contos, L. M. Ball, and J. Lewtas, Detection of Hydroxylated Nitro Aromatic and Hy-droxylated Nitro Polycyclic Aromatic Compounds in an Ambient Air Particulate Extract Using Bioassay-Directed Fractionation, Environ. Sci. Technol., 22, 908-915 (1988). 

Many starfish cause an escape response in usually sessile marine invertebrates [7]. The starfish Dermasterias imbricata causes the sea anemone Stomphia coccinea to release its basal disc from the substratum and swim away on contact. Bioassay-directed fractionation of the starfish extract led to the isolation of the compound found to elicit this response, the benzyltetrahydroisoquinoline alkaloid imbricatine (646). The structure of compound 646 was elucidated by spectral data interpretation. The amino acid residue in imbricatine is related to the thiol containing amino acids ovothiols A-C. Imbricatine (646) is active in both LI210 and P388... 

Bioassay-directed fractionation of this plant led to the isolation of the known alkaloid verazine (16) and eight related compounds (six of them newly reported) as the bioactive constituents alkaloids 17 and 18 are examples of the new compounds isolated. The relative stereochemistry of the new alkaloids was established by careful ID- and 2D-NMR experiments. 

Wilfordil Hook F (celastraceae) on the basis of bioassay-directed fractionation. The ethanol extract was concentrated in an ethylacetate layer of an ethylacetate-water participation. The ethylacetate extract was eluted on silica gel with chloroform and 5% methanol in chloroform. The latter fraction was further chromatographed on silica AR-CC-7 with chloroform to yield a triptolide-enriched fraction. This was further chromatographed on silica AR CC-7 to yield triptolide (137) which on crystallization from CH2Cl2-Et20 was obtained as white needles, m.p. 

Ajowan is known to traditional healers to have hypotensive properties. Bioassay-directed fractionation of seeds results in the isolation of thymol. In anaesthetized rats, thymol (1—lOmg/kg, i.v.) produces dose-dependent reductions in blood pressure and heart rate (Aftab et al., 1995). 

Environmental samples often contain swathes of different chemicals in mixtures. An important question for risk assessment, regulation, and remediation is to establish whether the majority of chemicals contribute to the overall mixture effect, or whether joint toxicity can be traced back to a few substances. This issue has been the topic of considerable research efforts in the field of ecotoxicology. Its resolution has required whole mixture approaches, where environmental samples were subjected to extraction procedures, followed by fractionation and chemical analysis (toxicity identification evaluation (TIE), bioassay-directed fractionations). There are interesting examples in the literature where such approaches were combined with component-based mixture assessments with the aim of identifying chemicals that contribute to mixture effects (see Chapter 4). 

Chief among the knowledge gaps that currently impede progress is a lack of information about cumulative exposure scenarios. Comparatively few studies have measured multiple chemicals in one and the same sample, and consequently, information about how many pollutants co-occur, and at what levels, is patchy. Viable concepts for cumulative exposure assessment strategies need to be developed, and the experiences that have occurred in the areas of bioassay-directed fractionations and with the toxicity identification evaluation (TIE) concepts no doubt provide valuable stimuli. 

BDF Bioassay-directed fractionation. Fractionation of mixture samples followed by toxicity testing of the fractions in a bioassay. Often performed to identify the most toxic fraction for further chemical characterization. 

Sezik E, Aslan M, Yesilada E, Ito S. Hypoglycaemic activity of Gentiana olivieri and isolation of the active constituent through bioassay-directed fractionation techniques. Life Sci 2005 76(ll) 1223-38. 

A bioassay directed fractionation of A. kurodai collected from Mei Prefecture in Japan has... 

A number of alkaloids producing a pronounced enhancement of phagocytosis were isolated by Wagner et al. (20) As a part of our continuing search for new biologically active metabolites from U. tomentosa we preceded to a bioassay-directed fractionation of the extracts from V. tomentosa. The extracts and fractions have been bioassayed by the carrageenan induced edema test on rat paw. This allowed us to identify a quinovic acid derivative with a C-3,27 glycosidation pattern, 21. as one of the active anti-inflammatory principles of U. tomentosa. (11). 

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