Induction assay

Safe, S. 1987b. Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs) support for the use of the in vitro AHH induction assay. Chemosphere 16 791-802. 

Houk VS, DeMarini DM. 1988. Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes. Environ Mol Mutagen 11 13-29. 

Jagannath DR. 1981. Mutagenic evaluation of S276 in the Saccharomyces cerevisiae reverse mutation induction assay. Study No. T4003065. Litton Bionetics Inc., Kensington, MD. 

Overall Evaluation of High-Throughput Induction Assays... 

Su, B.-N. et al. Activity-guided isolation of the chemical constituents of Muntingia calabura using a quinone reductase induction assay, Phytochemistry, 63, 335, 2003. 

Zacharewski T, Harris M, Safe S, et al. 1988. Applications of the in vitro aryl hydrocarbon hydroxylates induction assay for determining "2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents" Pyrolyzed brominated flame retardants. Toxicology 51 177-189. 

Thomson, J.A. (1981) Mutagenic activity of 42 coded compounds in the lambda induction assay. In de Serres, F.J. Ashby, J., eds. Progress in Mutation Research, Vol. 1, Evaluation of Short-Term Tests for Carcinogens. Report of the International Collaborative Program, Amsterdam, Elsevier/North-Holland, pp. 224-235... 

Water, soil (2,3,7,8-TCDD equivalents) Details for sample preparation were not reported by the authors. Enzyme induction assay (EROD) 62.5 pg/L No data Schuman and Hunter 1988... 

Cytochrome P450 Induction Assay in Primary Cultured Hepatocytes... 

Enzyme induction assays are generally performed for the evaluation of the effects of the substance tested on P450 induction. For animal studies, liver enlargement (e.g. liver weight gain) as well as increases in P450 activities after a relevant treatment period (e.g. 14 days) are used as endpoints for enzyme induction potential. 

The following represents the timeline and procedures for a typical enzyme induction assays with human hepatocytes (Table 3) ... 

Activity Determination (Day 6) The cells are harvested for the evaluation of enzyme activities. This can be performed in situ via the incubation of the cells with the enzyme substrates (Table 2). Activity can also be determined from the cell homogenates or microsomal fractions of the cells. The in situ method is the most efficient, and would allow one to use 24-well or 96-well plates. The microsomal method would require the use of larger cell culture plates (e.g. 100-mmdiameterplates). DME substrates used in the hepatocyte enzyme induction assay are shown in Table 4. 

Table 3 Timeline and key procedures for the human hepatocyte enzyme induction assay. Days Procedures Comments...

Appropriate statistical methods are yet to be established for enzyme induction assays. In general, ANOVA is used for the determination of statistical significance. Biological significance such as fold induction and effective concentrations (e.g. EC50) are valuable in the interpretation of the significance of the data. One interesting approach is to compare the data to that of the positive control which also allows the normalization of data for inter-experimental comparisons. 

Besides enzyme activity, enzyme induction assays can also utilize mRNA and enzyme protein level as endpoints. Gene expression studies now can be performed using branch-chained DNA and microarray techniques. Protein level quantification in general is performed using isoform-specific antibodies and Western blotting. Enzyme activity represents the most relevant endpoint for drug-drug interaction evalua-... 

Table 4 Drug metabolizing enzyme (P450 isoforms (CYP), UDP-glucuronosyl transferase (UGT) and phenol sulfotransferase (PST)) substrates that can be used for the in situ measurement of activity in the human hepatocyte enzyme induction assay.

Freshly isolated hepatocytes are universally accepted as the gold standard for enzyme induction assays. However, each experiment requires the availability of a liver for hepatocyte isolation. Fresh hepatocyte availability has been recently enhanced due to several commercial vendors effort to procure livers and provide isolated hepatocytes as a product. Studies with fresh hepatocytes cannot be planned, and sometimes may be delayed due to the lack of livers. To overcome this inconvenience of the use of freshly-isolated human hepatocytes, the following approaches for enzyme induction have been developed ... 

Many of the toxic and biological effects induced by polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and PCBs such as carcinogenesis, reproductive disturbances and immunotoxic effects are believed to be mediated via the hepatic cytosolic aryl hydrocarbon receptor (Ah receptor) [254,255]. Based on in vitro and in vivo studies, the toxicity of individual organochlorines have been determined relative to 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) and expressed as toxic equivalency factors (TEFs) [254, 256]. In addition to PCDD/F, structurally related PCBs and PCNs bind to the Ah receptor. After binding to the Ah-receptor, the receptor-ligand complex is transferred into the nucleus where it binds to specific DNA sequences and causes transcription of structural genes, which in turn causes synthesis of various cytochrome P4501A1-dependent enzymes such as ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH). TEFs for PCNs have been estimated from enzyme-induction assays of EROD and AHH [10, 257] and Luciferase assays in rat cells [12] cf. Table 4. 

Cytotoxic compounds Induction effects can be masked by the decrease of cell viability, as most induction assays quantify substrate metabolism in situ (in the same cell culture plate that the cells are cultured) and assume that there is no change in cell number. Cytotoxicity evaluation therefore should always be performed concurrently with induction studies. In the presence of cytotoxicity, activity should be corrected by the viability for comparison with negative control activity to assess induction potential. 

Mutagenesis tests of 2,6-xylidine have proved equivocal. o-Toluidine has displayed positive results in DNA repair assays and phage induction assays. The parent compounds displayed no mutagenicity. The mutagenesis potential has not been evaluated in the majority of local anesthetics. 

Thomson JA. 1981. Mutagenic activity of 42 coded compounds in the lambda induction assay. Prog Mutat Res 1 224-235. 

Using the Biological Induction Assay (BIA) and Micrococcus luteus... 

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