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in the urine and feces and/or in blood or other tissue. Again, the use of the intact ani-
mal has practical advantages over in vitro methods, although little is revealed about the
mechanisms involved.
Effects on in Vitro Metabolism Following in Vivo Treatment
This method of demonstrating inhibition is of variable utility. The preparation of
enzymes from animal tissues usually involves considerable dilution with the preparative
medium during homogenization, centrifugation, and resuspension. As a result, inhibitors
not tightly bound to the enzyme in question are lost, either in whole or in part, dur-
ing the preparative processes. Therefore, negative results can have little utility because
failure to inhibit and loss of the bound inhibitor give identical results. Positive results,
on the other hand, not only indicate that the compound administered is an inhibitor
but also provide a clear indication of excellent binding to the enzyme, most probably
due to the formation of a covalent or slowly reversible inhibitory complex. The inhibi-
tion of acetylcholinesterase following treatment of the animal with organophosphorus
compounds, such as paraoxon, is a good example, because the phosphorylated enzyme
is stable and is still inhibited after the preparative procedures. In contrast, inhibition
by carbamates is greatly reduced by the same procedures, because the carbamylated
enzyme is unstable and, in addition, the residual carbamate is diluted.
Microsomal monooxygenase inhibitors that form stable inhibitory complexes
with CYPs, such as SKF-525A, piperonyl butoxide and other methylenedioxyphe-
nyl compounds, amphetamine and its derivatives, and organophosphorus pesticides
(OPs) containing the P
S moiety, can be readily investigated in this way because the
microsomes isolated from pretreated animals have a reduced capacity to oxidize many
xenobiotics.
In Vitro Effects
In vitro measurement of the effect of one xenobiotic on the metabolism of another
is by far the most common type of investigation of interactions involving inhibition.
Although it is the most useful method for the study of inhibitory mechanisms, par-
ticularly when purified enzymes are used, it is of more limited utility in assessing the
toxicological implications for the intact animal. The principal reason for this is that in
vitro measurement of inhibition does not assess the effects of factors that affect absorp-
tion, distribution, and prior metabolism, all of which occur before the inhibitory event
under consideration and affect the concentration of inhibitor at the site of action.
The primary considerations in studies of inhibition mechanisms are reversibil-
ity and selectivity. The inhibition kinetics of reversible inhibition give considerable
insight into the reaction mechanisms of enzymes and, for that reason, have been well
studied. In general, reversible inhibition involves no covalent binding, occurs rapidly,
and can be reversed by dialysis or by dilution. Reversible inhibition is usually divided
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