A Practical Approach, Second Edition=Ronald D. Ho.pdf

532 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONacademic interest, such a finding may not be meaningful from a practical point of view because itmay not provide an adequate explanation for the toxicity of the chemical. Since the quantity ofproduct formed is determined by the amount of enzyme, it is important that the bioactivationenzyme occurs in the tissue in biologically significant amounts. Furthermore, the catalytic efficiencyof the enzyme must be high enough to generate an amount of ultimate toxicant adequate to interactwith target molecules and induce toxicity. This implies that the generation of ultimate toxicantmust overwhelm the endogenous defense and repair mechanisms. That thresholds exist and themajority of babies born today are healthy and normal despite everyday low exposures of womento many chemicals throughout pregnancy attests to this contention. In this section, assay proceduresare given for estimating activities of major xenobiotic metabolizing enzymes.B. Assay Methods1. Cytochrome P450 EstimationThe most commonly used method is that of Omura and Sato, 38 which is based on the reducedcarbon monoxide (CO) difference spectrum.a. Reagents1. 0.1 M potassium phosphate buffer, pH 7.42. Sodium dithionite, reagent grade3. CO gas, reagent grade (use in fume hood)b. ProcedureWashed microsomes in the buffer (1 to 3 mg protein/ml) are placed in two matching cuvettes (1.0-cm light path). By use of a spectrophotometer equipped with a turbid sample accessory operatedin split beam mode, a baseline of equal absorption is recorded between 400 and 500 nm. Thecontents of the sample cuvette are saturated with CO (one bubble per second for about 30 sec). Afew crystals (about 1 mg) of sodium dithionite are added to both cuvettes. The cuvettes are coveredwith Parafilm ® , and the contents are gently mixed by inverting the cuvettes three or four times.Difference spectra are then recorded (400 to 500 nm) until absorbance at the 450-nm peak reachesmaximum. P450 content is determined using the following equation:[(A450 to 490 nm) observed (A450 to 490 nm) baseline]/0.091 = nmol P450/milliliterc. RemarkIn the case of significant hemoglobin (Hb) contamination, the procedure of Matsubara et al. 39 isrecommended.2. Cytochrome P450–Mediated Xenobiotic OxidationXenobiotic oxidation observed in a reconstituted system containing purified P450 enzyme and/orinhibition of the reaction by the inclusion of antibody to NADPH P450 reductase provide strongevidence for the involvement of P450. The results of experiments using preferred substrates incombination with selective inhibitor(s) and a specific antibody can provide useful data on thepossible contribution of individual P450 enzymes to xenobiotic oxidation in microsomes. P450 cancatalyze a wide spectrum of xenobiotic oxidation reactions. For each type of reaction, a choice ofsubstrate is available. Typical assay conditions employed in such experiments are presented below© 2006 by Taylor & Francis Group, LLC