Search Menu Abstract The combination effects of the well-known antiandrogenic fungicides, vinclozolin and procymidone, were tested both in vitro and in vivo.
By applying the isobole method, the effect of combining the two pesticides in vitro was found to be additive. Using the isobole method, comparisons of the observed and predicted effects assuming additivity on reproductive organ weights, hormone levels, and gene expression showed agreement and thus the combination effects are suggested to be additive in vivo as well as in vitro.
Many compounds have been shown to be antiandrogenic in vitro and in animal studies Gray et al. Based on the effect of single compounds, it is difficult to explain the possible impact on human health.
Very little is known on the human exposure to these compounds, but it is anticipated that humans are exposed to a mixture of many different endocrine disrupters from different sources, each present at low levels Wilkinson et al. Thus, it is highly relevant to get more knowledge on potential combination effects of similarly acting compounds and investigate this issue thoroughly in diverse model systems.
One work by Arnold et al. Some studies on combinatorial effects have only tested one dose level of each compound alone and in combination. In these studies it was predicted that the combination effects followed effect summation at all concentration levels, thus a linear dose-response curve with a plot passing through the origin was assumed.
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Linear dose-response curves can be found in the low dose range, e. As the linearity was not demonstrated in most of the combination studies mentioned above, they were found to be inconclusive when evaluated thoroughly Kortenkamp and Altenburger, In most studies, additivity and thus no interaction of the combination of compounds was observed, but a thorough evaluation of the results showed that synergism had been overlooked in a few cases Kortenkamp and Altenburger, The best fitted regression lines for the observed effects of the mixtures were congruent with the curves for the predicted additive effect.
By calculated predictions and concentration-response curves they found the combination of the estrogenic compounds to be additive.
Payne et al. The combined effect was found additive by this approach. Recently, Rajapakse et al.
Most of the published studies concerning mixtures of endocrine disrupters have looked at the effect of combination of the compounds in vitro. Interactions with the receptor in vitro are often found to be additive Payne et al. Adding to the complexity, it has been shown that combination effects can vary with different endpoints within the same experimental design.
Compounds working synergistically on the molecular level can elicit additivity on more complex endpoints such as cell death Grimme et al. In this study, the combination of two similarly acting fungicides was investigated in vitro and in vivo with endpoints at the molecular as well as the organ level.
Dose-response curves of both fungicides known to elicit antiandrogenic effects and mixtures of these were analyzed in an in vitro androgen reporter gene normal size prostate gland ml Vinggaard et al. The Hershberger assay was supplemented with hormone analyses as well as with quantitative analyses of androgen-dependent gene expression as previously described Nellemann et al.
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- No treatment related changes were considered for number of still births and runts.
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Few studies of endocrine disrupters so far have included evaluation of neurochemical parameters. Following analyses of the dose-response curves for the single fungicides and assuming additivity, the models of concentration addition were used to predict the relationships for combination of the two with a variety of fixed mixture ratios.
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Testosterone propionate CAS no. F from Sigma-Aldrich St. Louis, MO. Vinclozolin CAS no.
AR reporter gene assay. Effects on androgen receptor activity were tested in a previously developed reporter gene assay Vinggaard et al. The chemicals to be tested were added with or without 0.
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Hydroxyflutamide was included in every experiment as a positive control. Luciferase activity was measured directly using a BioOrbit Galaxy luminometer by automatically injection of 40 μl substrate containing 1 mM luciferin Amersham Int. The observations were normalized to correct for interassay variation.
The procedure is described in detail in Andersen et al. Animal Experiments Test species. All rats were castrated at the age of four weeks, 14 days prior to study start.
They were fed Normal size prostate gland ml 8.