A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations.
Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell-free approach, this method is unreliable for cell or tissue-based investigations as other cytochrome P-450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose-related inhibitions when incubating non-pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane. Additional cytochrome P-450s were also measured. Treatment-related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme.
Bayer SAS, Bayer CropScience, Research Toxicology, Sophia Antipolis, France. helen.tinwell@bayer.com
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Tinwell
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Rascle
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Colombel
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Al Khansa
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Freyberger
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Bars
D000826:Animal Testing Alternatives; D000818:Animals; D001141:Aromatase / Q000032:analysis* / Q000187:drug effects; D004796:Clinical Enzyme Tests / Q000379:methods*; D003632:Dichlorodiphenyldichloroethane / Q000633:toxicity; D052244:Endocrine Disruptors / Q000494:pharmacology; D004784:Environmental Monitoring / Q000379:methods; D004958:Estradiol / Q000032:analysis / Q000378:metabolism*; D005260:Female; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D010053:Ovary / Q000187:drug effects* / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000378:metabolism / Q000494:pharmacology*; D014316:Tritium / Q000032:analysis / Q000737:chemistry; D014867:Water / Q000737:chemistry
D052244:Endocrine Disruptors; D014867:Water; D014316:Tritium; D013739:Testosterone; D004958:Estradiol; D001141:Aromatase; D003632:Dichlorodiphenyldichloroethane