The combined effects of vinclozolin and procymidone do not deviate from expected additivity in vitro and in vivo.

Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, Mørkhøj Bygade 19, Dk-2860 Søborg, Denmark.
Toxicological Sciences (Impact Factor: 3.85). 03/2003; 71(2):251-62.
Source: PubMed


The combination effects of the well-known antiandrogenic fungicides, vinclozolin and procymidone, were tested both in vitro and in vivo. In vitro both vinclozolin and procymidone significantly inhibited the binding of agonist to the androgen receptor with the concentration that resulted in 50% inhibition (IC(50)) values of 0.1 and 0.6 micro M, respectively. By applying the isobole method, the effect of combining the two pesticides in vitro was found to be additive. In castrated testosterone-treated rats the administration of vinclozolin starting at 10 mg/kg led to a decrease in organ weight of all tested reproductive organs. The levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were increased significantly with doses of 100 mg/kg vinclozolin and above. Expression of the androgen-responsive gene, TRPM-2, was increased starting at 100 mg/kg vinclozolin. For procymidone, reproductive organ weights were diminished at 10 mg/kg and LH was increased at a concentration of 25 mg/kg and above, compared to the testosterone-treated controls. FSH was significantly increased only at 25 mg/kg procymidone. The studied gene expressions were changed by 100 mg/kg procymidone. Dosing the animals with a combination of a 1:1 mixture of vinclozolin and procymidone resulted in a weight reduction in the reproductive organs and an increase of serum LH and FSH as early as with 10 mg/kg combined dose. The relative expressions of TRPM-2 and PBP C3 were changed compared to controls at 100 mg/kg. The level of 5-HT in the rat brain was increased after a dose of 10 mg/kg. 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.

Download full-text


Available from: Henrik Rye Lam,
  • Source
    • "Considering that risk assessment procedures do not take account of mixture effects at present, it is possible that risks to male reproductive health by pesticides are being underestimated. Although antiandrogenic mixture effects have been described for certain pesticides, some of them obsolete (Nellemann et al. 2003, Kjaerstad et al. 2010, Birkhoj et al. 2004), similar data with more widely used pesticides are lacking. Since it is known that many current-use pesticides are AR antagonists in vitro (Kojima et al. 2004; Orton et al. 2009; Orton et al. 2011), it is plausible to assume that there might also be mixture effects of these pesticides. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: Many pesticides in current use have recently been revealed as in vitro androgen receptor (AR) antagonists, but information about their combined effects is lacking. Objective: We investigated the combined effects and the competitive AR antagonism of pesticide mixtures. Methods: We used the MDA-kb2 assay to test a combination of eight AR antagonists that did not also possess AR agonist properties (“pure” antagonists; 8 mix: fludioxonil, fenhexamid, ortho-phenylphenol, imazalil, tebuconazole, dimethomorph, methiocarb, pirimiphos-methyl), a combination of five AR antagonists that also showed agonist activity (5 mix: cyprodinil, pyrimethanil, vinclozolin, chlorpropham, linuron), and all pesticides combined (13 mix). We used concentration addition (CA) and independent action (IA) to formulate additivity expectations, and Schild plot analyses to investigate competitive AR antagonism. Results: A good agreement between the effects of the mixture of eight “pure” AR antagonists and the responses predicted by CA was observed. Schild plot analysis revealed that the 8 mix acted by competitive AR antagonism. However, the observed responses of the 5 mix and the 13 mix fell within the “prediction window” boundaries defined by the predicted regression curves of CA and IA. Schild plot analysis with these mixtures yielded anomalous responses incompatible with competitive receptor antagonism. Conclusions: A mixture of widely used pesticides can, in a predictable manner, produce combined AR antagonist effects that exceed the responses elicited by the most potent component alone. Inasmuch as large populations are regularly exposed to mixtures of antiandrogenic pesticides, our results underline the need for considering combination effects for these substances in regulatory practice.
    Environmental Health Perspectives 09/2012; 120(11). DOI:10.1289/ehp.1205391 · 7.98 Impact Factor
  • Source
    • "The obtained effects of the mixtures were interpreted as to represent dose-additivity. Results of in vitro and in vivo combination experiments with procymidone and vinclozolin were also interpreted as to be in agreement with expected dose additivity (Nellemann et al., 2003). In this study we have assessed the hAR mediated antiandrogenic effects of binary mixtures of the two antiandrogens flutamide and vinclozolin in the presence of the androgen DHT in a yeast based transcriptional activation assay. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Humans are exposed to a combination of various substances such as cosmetic ingredients, drugs, biocides, pesticides and natural-occurring substances in food. The combined toxicological effects of two or more substances can simply be additive on the basis of response-addition, or it can be greater (synergistic) or smaller (antagonistic) than this. The need to assess combined effects of compounds with endocrine activity is currently discussed for regulatory risk assessment. We have used a well described yeast based androgen receptor transactivation assay YAS to assess the combinatorial effects of vinclozolin and flutamide; both mediating antiandrogenicity via the androgen receptor. Both vinclozolin and flutamide were antiandrogens of similar potency in the YAS assay. In the concentration range tested the two antiandrogens vinclozolin and flutamide did not act synergistically. Concentration additivity was observed in the linear, non-receptor-saturated concentration range. At high concentrations of one of the two substances tested the contribution of the second at lower concentration levels was less than additive. The combined response of both compounds at high concentration levels was also less than additive (saturation effect). At concentration levels which did not elicit a response of the individual compounds, the combination of these compounds also did not elicit a response.
    Regulatory Toxicology and Pharmacology 05/2011; 60(3):373-80. DOI:10.1016/j.yrtph.2011.05.005 · 2.03 Impact Factor
  • Source
    • "This conclusion is supported by the work of others. Gray et al. (2001) and Nellemann et al. (2003) found that mixtures of vinclozolin and procymidone resulted in dose additive decrease in androgensensitive organ weights, androgen levels, and AR-dependent gene expression in castrated, testosterone-treated male rats. Birkhoj et al. (2004) assessed the in vitro and in vivo effects of a combination of five pesticides with dissimilar mechanisms of action and found androgen-sensitive endpoint responses greater than would be expected using response addition modeling. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act required the US Environmental Protection Agency to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures of chemicals to elucidate mechanisms of joint action at the systemic level with the goal of providing a framework for assessing the cumulative effects of reproductive toxicants. Previous mixture studies conducted with antiandrogenic chemicals are reviewed briefly and two new studies are described. In all binary mixture studies, rats were dosed during pregnancy with chemicals, singly or in pairs, at dosage levels equivalent to approximately one-half of the ED50 for hypospadias or epididymal agenesis. The binary mixtures included androgen receptor (AR) antagonists (vinclozolin plus procymidone), phthalate esters [di(n-butyl) phthalate (DBP) plus benzyl n-butyl phthalate (BBP) and diethyl hexyl phthalate (DEHP) plus DBP], a phthalate ester plus an AR antagonist (DBP plus procymidone), a mixed mechanism androgen signalling disruptor (linuron) plus BBP, and two chemicals which disrupt epididymal differentiation through entirely different toxicity pathways: DBP (AR pathway) plus 2,3,7,8 TCDD (AhR pathway). We also conducted multi-component mixture studies combining several 'antiandrogens'. In the first study, seven chemicals (four pesticides and three phthalates) that elicit antiandrogenic effects at two different sites in the androgen signalling pathway (i.e. AR antagonist or inhibition of androgen synthesis) were combined. In the second study, three additional phthalates were added to make a 10 chemical mixture. In both the binary mixture studies and the multi-component mixture studies, chemicals that targeted male reproductive tract development displayed cumulative effects that exceeded predictions based on a response-addition model and most often were in accordance with predictions based on dose-addition models. In summary, our results indicate that compounds that act by disparate mechanisms of toxicity to disrupt the dynamic interactions among the interconnected signalling pathways in differentiating tissues produce cumulative dose-additive effects, regardless of the mechanism or mode of action of the individual mixture component.
    International Journal of Andrology 04/2010; 33(2):443-62. DOI:10.1111/j.1365-2605.2009.01049.x · 3.70 Impact Factor
Show more