Estimation of estrogenic and antiestrogenic activities of selected pesticides by MCF-7 cell proliferation assay.

Estrogenic activities of 20 selected pesticides-which are used for agricultural production as insecticides, fungicides and herbicides-were examined by estrogen receptor (ER)-dependent MCF-7 cell proliferation assay. Among them, chlordecone, dicofol, methoxychlor, gamma-HCH, fenarimol, EPN, triadimefon, and triadimenol had estrogenic activities, all of which were suppressed by the addition of pure antiestrogen ICI 182,780. The first 5 compounds exhibited binding capacities to ERalpha. The antiestrogenic activity of a compound was examined by estimating its suppressive effect on cell proliferation induced by 30 pM 17beta-estradiol. Strongly suspected antiestrogens were captan and myclobutanil, both of which were found to have the capacity to bind to ERalpha and which might exert their activities by competing at the level of ERalpha. Antiestrogenic activities of nitrofen, fenitrothion, fenarimol and triadimefon were also suggested. Affinities of the compounds for ERalpha and/or androgen receptor (AR) were lower than those of synthetic estrogen (diethylstilbestrol) and testosterone (mibolerone), respectively. Fenitrothion had the highest affinity to AR. Chlordecone, dicofol, methoxychlor, nitrofen, fenarimol, myclobutanil and pyridate had capacities to bind both ERalpha and AR. Chlordecone and pyridate were much more effective as competitors of estrogen binding to ERalpha than androgen binding to AR and, conversely, nitrofen was a more effective competitor of androgen binding to AR.

Kinetic assay of serum and urine for urea with use of urease and leucine dehydrogenase.

We describe a new kinetic assay for determining urea in serum or urine with use of urease (EC 3.5.1.5) and leucine dehydrogenase (EC 1.4.1.9). The latter enzyme is suitable for the kinetic assay of NH4+ because its Km value for NH4+ at pH 8.75 is large (approximately 500 mmol/L). Interference from endogenous NH4+ in serum or urine is obviated by subtraction of the assayed endogenous NH4+ value in a sample blank. For serum, within-assay CVs (n = 10) were 0.39-0.58%; day-to-day CVs (n = 10) were 1.56-2.30%. In urine, within-assay CVs (n = 10) were 0.86-1.15%. Analytical recovery of urea (0.893-71.4 mmol/L) added to patients' sera (urea 6.14 mmol/L) was 99.2-105.2%. The calibration curve for serum was linear through zero for urea concentrations up to 142.9 mmol/L and for urine up to 714.3 mmol/L. No influences of added ammonium ion, bilirubin, hemoglobin, ascorbic acid, or Intralipid were observed. The regression equations for this method (y) and conventional methods (x = Determiner-LUN for serum assays, Serotec UUR-R for urine) were: y = 1.016x - 0.12 mmol/L (r = 0.999, S(y/x) = 0.34 mmol/L, n = 100) for sera, and y = 1.070x - 12.6 mmol/L (r = 0.998, S(y/x) = 7.41 mmol/L, n = 100) for urine.