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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, volume prostata 25 ml 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.

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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.

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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. 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 volume prostata 25 ml 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.

Neonatal brain damage Abstract A specialized neurogenic niche along the ventricles accumulates millions of progenitor cells in the developing brain.

Payne et al. The combined effect was found additive by this approach. Recently, Rajapakse et al.

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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 assay Vinggaard et al. The Hershberger assay was supplemented with hormone analyses as well as with volume prostata 25 ml analyses of androgen-dependent gene expression as previously described Nellemann et al.

Few studies of endocrine disrupters so far have included evaluation of neurochemical parameters.

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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. 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. Hydroxyflutamide was included in every experiment as a positive control. Volume prostata 25 ml 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 Syn 8.

ABHD4-dependent developmental anoikis safeguards the embryonic brain | Nature Communications

IT a diet known to be free of phytoestrogens and were provided with acidified tap water ad libitum. Rats were weighed and divided by randomization into treatment groups. During testing rats were weighed daily and visually inspected for health effects twice a day.

Testing of vinclozolin and procymidone in castrated animals. In Experiment 1 all groups of male castrated Wistar rats seven groups of six animals each were administered 0.

In Experiment 2 all groups of male castrated Wistar rats seven groups of six animals each were administered 0. In Experiment 3 all groups of male castrated Wistar rats nine groups of six animals each were administered 0. For all three experiments all compounds were dissolved in peanut oil. Sterile oil The Royal Veterinary Agriculture Pharmacy, Copenhagen, Denmark was used for testosterone propionate and flutamide solutions. The testosterone dose was always given a few minutes after the test compound and the last dosing was performed in the morning on the day of killing the animals.

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All the animals from each group underwent a thorough autopsy. The ventral prostates were stored in 0.

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Blood was collected in plain glass tubes and serum was prepared and stored at —80°C until measurement of hormones. Organon was used.

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Finally, europium-labeled streptavidin was bound to the biotin-labeled antibody. Buffers and washing solutions used throughout the assay were Delfia® reagents obtained from Wallac. The quantity and quality of the purified RNA was evaluated by spectrophotometry. Quantitative results were obtained by the cycle threshold value where a signal rises above background level.

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For TRPM-2 0. Gene expressions were analyzed at least three times for each animal in the same cDNA preparation. Neurochemistry Preparation of brain samples. The whole brain was isolated, EDF- terápia prosztatitis, and homogenized in 8 ml ice-cold 0.

Samples of homogenate were taken and processed for protein, enzyme, and neurotransmitter analyses. Protein analysis.

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A sample of homogenate was added to 0. Determinations of enzyme activities. After 1 h at ambient temperature, samples were centrifuged at 10, × g for 10 min.

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Neurotransmitter analyses. Immediately after homogenization, one part of a 0.

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One aliquot of perchloric acid supernatant Lam et al. Isobole method. In order to calculate a function describing the predicted effect of a combination of the compounds, additivity was assumed. The concentrations of the single compounds resulting in a given effect were calculated from the regression line giving the best fit of the concentration-response curve for each fungicide.

Statistical analyses.

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All data was analyzed by comparing the group treated with testosterone control and the groups treated with testosterone and flutamide or testosterone and vinclozolin or procymidone alone or in combination. A t-test was performed to compare the flutamide-treated animals with the controls.

IC50 values of the in vitro data were determined using a four-parameter logistic function SigmaPlot ver. Vinclozolin alone inhibited the response induced by 0.

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At concentrations above 3 μM and up to 50 μM, vinclozolin showed agonistic activity. This is in agreement with a previous report that showed agonism at higher concentrations of vinclozolin Wong et al.

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Procymidone alone showed competitive binding to the androgen receptor at all concentrations studied Fig. The IC50 of vinclozolin and procymidone was determined from the nonlinear regression line to be 0.

The combination studies of vinclozolin and procymidone were performed with, and mixtures Fig. The data from the experiments were fitted to a four-parameter logistic curve fit. Based on the fitted curves for the single compounds, the predicted effects assuming additivity of the combinations were calculated. The predicted additive effect of the mixture volume prostata 25 ml shown in Figure 2A as a dotted line.

The actual observations of combinations of vinclozolin and procymidone are shown as points with standard deviations. As depicted in Figure 2Athe combination of the two pesticides showed that vinclozolin and procymidone acted additively in combination and thus did not seem to interact in vitro.


The fitted curves and observations of the and mixes also showed additivity of the combination of the two compounds Figs. In vivo, all the reproductive organs investigated showed decreasing weights with increasing vinclozolin concentrations in the first experiment Table 1. Flutamide was used as positive control for the antiandrogenic effects and showed effect on all reproductive organs. No effect of treatment on body weight, liver weight, paired kidney weight, or pituitary weight was Prostatitis poliuria. As the body weights did not differ significantly between the groups the absolute organ weights are shown Table 1.

In the second experiment with increasing doses of procymidone, all the reproductive organs investigated showed decreasing weight with increasing procymidone concentration Table 2. The reason for that may be that only three prostates due to a technical error were available in most groups.

The chosen doses of procymidone did not have any effect on body weight, liver weight, paired kidney weight, or pituitary weight. As the body weights did not differ significantly volume prostata 25 ml the groups, the absolute organ weights are shown Table 2.

Increasing doses of the combination of vinclozolin and procymidone caused decreasing weight of the reproductive organs. The combination did not have any effect on body weight, liver weight, paired kidney weight, or pituitary weight. As the body weights did not differ significantly between the groups, the absolute organ weights are shown. The decrease in reproductive organ weights were normalized to the testosterone group and described by fitting the data to a four-parameter logistic curve fit.

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The classical isobole method on the changes in reproductive organ weights resulted in isobole coefficients of 0. These values below 1 may point to a synergistic effect of the combination of the two pesticides. But as depicted in Figs. The hormone analyses showed that both vinclozolin and procymidone are capable of increasing the level of LH and FSH.

In this experiment the flutamide administration did not result in any increase in FSH either. The treatment with a mixture of vinclozolin and procymidone treatment resulted in changes volume prostata 25 ml FSH levels significantly different from the control group for all treatment groups data not shown.

Isobole calculations showed that the combined effects of vinclozolin and procymidone did not differ from additivity at the hormone level data not shown.

The second experiment with procymidone did not give any useful observations, probably because only three prostates were available in most groups data not shown. The positive control, flutamide, gave significant results in all three experiments for both gene expressions.

Isobole calculations showed that the combined effects of vinclozolin and procymidone did not deviate from additivity at the gene expression level data not shown. In contrast, 5-HT data showed a statistically significant increase between the mean values of the different groups.

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The values for the flutamide group were compared to the testosterone group and were not found to be significantly different from the testosterone group. In the current study, each fungicide was studied thoroughly alone and in combination both in Prosztata kenőcs and in vivo in order to be able to evaluate whether any interaction occurred between the fungicides. Initially, the combination effects of procymidone and vinclozolin were tested in an AR reporter gene assay.

This is to our knowledge the first combination experiment on endocrine disrupters performed on AR in vitro. The assay is very useful for this purpose because it allows dose-response curves of the compounds alone and in several mixture ratios to be tested simultaneously. The results obtained showed that the isobole coefficients were 0.

In general, all results indicated unambiguously that the blocking effect on AR did not deviate from additivity. Volume prostata 25 ml vitro, procymidone showed a lower inhibitory effect than vinclozolin with an IC50 of 0.


This is in contrast to findings in an in utero study in Long-Evans rats treated from gestational day 14 to postnatal day 3 by Ostby et al. In intact adult male rats, Hosokawa et al.

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The current study shows that the potencies of androgen receptor antagonists in vitro do not always predict the potency of the compounds in vivo. One reason for the strong potency of procymidone in vivo might be that metabolites of procymidone act as antiandrogens.

In addition, the study shows that the Hershberger assay using young castrated male rats is a sensitive assay for evaluating the antiandrogenic effects of both procymidone and vinclozolin. The two pesticides were administered orally because the metabolites of vinclozolin, M1 and M2, are the active androgen receptor inhibitors and because human exposure is primarily oral Ashby and Lefevre, ; Kelce et al.