Preincubation Time

Preincubation of bacteria, S-9 Mix, and test substances greatly enhances the mutagenic potentials of many chemicals, and preincubation is essential for demonstrating the mutagenicities of some chemicals. 

FIGURE 4. Effects of the preincubation time on the mutagenicities of DMN and clivorine, a pyr-rolizidine alkaloid, on strain TAIOO. (a) DMN (3.7 mg) was incubated with bacteria and S-9 Mix from the liver of PCB-treated rats at 25°C before plating. Water was used as solvent, (b) Clivorine (1.5 mg) in DMSO was incubated with bacteria and S-9 Mix from the liver of PCB-treated rats at 37 C before plating. A correction was made for spontaneous revertants.  

FIGURE 5. Effects of preincubation on the mutagenicity of aflatoxin Bi (a), B[fl]P (b), and quinoline (c). Strains TAIOO (a, b) and TA98 (c) were used with the preincubation method ( ) and the pour-plate method (O). 

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Figure 6.2 Effect of preincubation time with inhibitor on the steady state velocity of an enzymatic reaction for a very slow binding inhibitor. (A) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the single-step binding mechanism of scheme B of Figure 6.3. (B) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the two-step binding mechanism of scheme C of Figure 6.3. Note that in panel B both the initial velocity (y-intercept values) and steady state velocity are affected by the presence of inhibitor in a concentration-dependent fashion.

Note that for very slow binding inhibitors that are studied by varying preincubation time, the fits of the exponential decay curves to Equation (6.4) provide values for both V, and kohs for each inhibitor concentration. The values of v, at each inhibitor concentration represent the v-intercepts of the best fit to Equation (6.4), and these can be used in conjunction with Equation (6.8) to obtain an independent estimate of... 

Figure 6.19 Fractional velocity for the enzymatic reaction of COX2 as a function of preincubation time with varying concentrations of the slow binding inhibitor DuP697. The lines drawn through the data represent the best fits to Equation (6.4).

This procedure can be modified to provide optimum conditions for particular chemical classes. For instance, preincubation times greater than 60 min plus aeration have been found necessary in the detection of allyl compounds (Neudecker and Henschler, 1985). 

Figure 9.12 Plots for interpreting TDI. (a) Natural log of percent activity remaining versus preincubation time (b) percent activity relative to control versus preincubation time (provides clarity regarding amount of competitive inhibition at chosen inhibitor concentrations ...

It is recommended that time-dependent inhibition be examined when deemed appropriate. Time-dependent inhibition should be examined with and without NADPH over an inhibitor concentration range of 1- to 10-fold the clinically relevant plasma concentrations. Various preincubation time points, such as 0, 15, 30, 45, and 60 minutes, should be utilized along with at least a 10-fold dilution step prior to the substrate incubation. 

Preincubation time with inhibitor Microsomal protein concentration ... 

The ratio of the preincubation time with inhibitor to the incubation time with maker substrate... 

Thus, primary plots of ln(% activity/100) versus t will be straight lines with slopes = —k as shown in Figure 57.7A. In addition, the experimentally determined dependence of % activity remaining on the preincubation time (t) and inhibitor concentration [OP] is... 

Reduce preincubation time to minimize loss of cytosolic proteins. Test the response of cells directly after SLO permeabilization. In contrast to Ca, the response to GTP[S] should not be altered by rundown (Kiraly-Borri efal., 1996). 

For mechanism-based inhibitors, is estimated by an experiment with varying inhibitor concentration and preincubation time. A typical fCinact study is as follows ... 

Preincubation time (preincubation of enzyme with inhibitor) Five (e.g., 5,10, 15, 20, 30 minutes)... 

Employment of glutamate oxidase enables the sequential assay of both transaminases without the need to coimmobilize a second enzyme. Using a glutamate oxidase sensor, Yamauchi et al. (1984) found a preincubation of the sample of 30 min to be necessary. The preincubation time could be lowered to 10 min by using an optimized sensor configuration (Wollenberger et al., 1989). 

The apparent inactivation rate constants of the enzyme, can be estimated from a series of experiments in which each of several concentrations of inhibitor [/] are used. For each [/] the slope of enzyme activity versus preincubation time is measured. The array of kot,s values is then plotted against inhibitor concentrations [/]. The Kiappis the [/] that elicits a ki aa/2. Graphic representations of these experiments are shown in Figure 8.29. 

In comparison with the reversible enzyme inhibition, the MBI can be characterized to be (1) dose-dependent (2) preincubation time-dependent (3) bioactivation required for inactivation of the target enzyme (4) de novo protein synthesis to return metabolic capacity and (5) slow onset of the effects but more profound than the reversible inhibition. 

Mechanism-based inhibition of the drug-metabolizing isoenzyme CYP3A4 was observed in human liver microsomes treated with the compounds rutaecarpine or limonin, with activity reported as preincubation time dependent (Iwata et al. 2005). 

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