Enzyme induction assay

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... 

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. 

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

Oral absorption of carbamazepine is quite slow and often erratic. Its half-life is reported to vary from 12 to 60 hours in humans. The development of blood level assays has markedly improved the success of therapy with this drug, since serum concentration is only partially dose related. Carbamazepine is metabolized in the liver, and there is evidence that its continued administration leads to hepatic enzyme induction. Carbamazepine-10,11-epoxide is a pharmacologically active metabolite with significant anticonvulsant effects of its own. 

D. C. Evans, D. P. Hartley, R. Evers (2001). Enzyme induction—Mechanisms, assays, and relevance to drug discovery and development. Ann. Rep. Med. Chem. 38 315. 

The assay of enzyme induction at the mRNA level is much easier to perform than the chemical assay of each individual enzyme and the response to a physiological event is intrinsically faster to detect. Moreover, mRNA is often more stable under cell extract preparation conditions than enzymes, if destruction by RNAses can be prevented. For analysing a number of different enzymes in parallel, only one sample preparation procedure is necessary, which produces a crude RNA extract, and only in the final step the different DNA or RNA probes are used for detection of specific enzyme RNAs. Recently, the detection of RNA is further facilitated by ready-to-use RNA isolation kits, non-radioactive DNA or RNA probes and the dot-blot and slot-blot techniques, respectively, [51,52]. 

Cumulative plots of carbofuran mineralization in a history and a nonhistory soil are presented in Figure 1. The initially accelerating rate of C02 production, indicative of a microbial response (e.g., enzyme induction, population growth), is characteristic of enhanced degradation. Comparison of the mineralization of u-carbonyl and 14C-ring labelled carbofuran demonstrates an important consideration in this type of assay the location of the u label in the insecticide is critical for this type of assay to provide useful information. Although the carbonyl C was almost completely evolved as CO2, the ring C was only slowly mineralized. 

When the enzyme-treated preparations were tested for biological activity in the wilt induction assay it was found that the Rhizopus enzyme-treated preparation had lost its biological activity (Figure 4). The Bacillus enzyme-treated preparation retained its activity as might be expected from the inability of this enzyme to depolymerise the preparation. After treatment with the Euglena exo-hydrolase, which caused only partial degradation, the polymer retained most of its wilt-inducing activity. 

Judged by enzyme induction, Menary and Jones (1972) indicated that nitrate was unable to move from a storage pool (vacuole) to the cytoplasm of ripe paw paw fruit. Using a modified in vivo assay, Ferrari et al. (1973) estimated sizes of active (metabolic) and inactive (storage) pools of nitrate in tobacco cells, barley aleurone layers and maize leaves. The location and nature of the pools was not defined. 

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