Quantification of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activity in human placentae: development of a protocol suitable for studying effects of environmental exposures on human metabolism

Several issues regarding measurement of placental AHH and 7ECD activity were studied, and standardized procedures that appeared more suitable than previous assay procedures for measurement of MFO induction in epidemiological studies were adopted. In the AHH assay, deletion of the rat-liver supernatant eliminated a possible extraneous contribution to measurement of low levels of AHH activity and did not substantially affect measurement of higher levels of activity. Increasing the protein concentration of placentae homogenate from 2 to 6 mg, and the length of the incubation time from 20 to 60 min, allowed for accumulation of more BaP products, potentially maximizing the detection of low levels of AHH activity. Use of tissue homogenates made the procedure more convenient and did not appear to interfere with interindividual comparisons of activity. Assay of homogenates of fresh and frozen tissue from the same placenta gave similar results, so that frozen tissue was adopted for convenience and replicability. Although a potential problem for specimens with high AHH activity, degradation of product(s) was modest in AHH assays of human placenta. The efficiency of extraction of fluorescent products declined with increasing protein concentrations in the reaction mixture of AHH assays, but it was stable for a range of product concentrations, and could be controlled by the use of a constant amount of protein per assay. Recovery of product for the 7ECD assay was more complete and was not affected by protein concentration. Additionally, the 7ECD activity was easily detected in every placenta, regardless of smoking exposure. However, in this study the AHH assay appeared to be better at discriminating between non-smokers and smokers. These observations, and the potential differences in the spectrum of agents causing induction of mixed-function oxidases, suggest that both assays are potentially useful measures of human MFO induction in clinical or epidemiological studies.