Systemic administration of diarylpropionitrile (DPN) or phytoestrogens does not affect anxiety-related behaviors in gonadally intact male rats

Abstract

The development of highly selective agonists for the two major subforms of the estrogen receptor (ERalpha and ERbeta) has produced new experimental methodologies for delineating the distinct functional role each plays in neurobehavioral biology. It has also been suggested that these compounds might have the potential to treat estrogen influenced behavioral disorders, such as anxiety and depression. Prior work has established that the ERbeta agonist, diarylpropionitrile (DPN) is anxiolytic in gonadectomized animals of both sexes, but whether or not this effect persists in gonadally intact individuals is unknown. Isoflavone phytoestrogens, also potent but less selective ERbeta agonists, have also been shown to influence anxiety in multiple species and are becoming more readily available to humans as health supplements. Here we determined the effects of 0.5, 1 or 2 mg/kg DPN, 1 mg/kg of the ERalpha agonist propyl-pyrazole-triol (PPT), 3 or 20 mg/kg of the isoflavone equol (EQ) and 3 or 20 mg/kg of the isoflavone polyphenol resveratrol (RES) on anxiety behavior in the gonadally intact male rat using the light/dark box and the elevated plus maze. We first determined that DPN can be successfully administered either orally or by subcutaneous injection, although plasma DPN levels are significantly lower if given orally. Once injected, plasma levels peak rapidly and then decline to baseline levels within 3 h of administration. For the behavioral studies, all compounds were injected and the animals were tested within 3 h of treatment. None of the compounds, at any of the doses, significantly altered anxiety-related behavior. Plasma testosterone levels were also not significantly altered suggesting that these compounds do not interfere with endogenous androgen levels. The results suggest that the efficacy of ERbeta agonists may depend on gonadal status. Therefore the therapeutic potential of ERbeta selective agonists to treat mood disorders may be limited.

Fig. 1

Time course of plasma DPN levels following a single injection of 1 mg/kg bw. Samples (one per animal; Wistar males) were collected over a five hour window beginning one hour after the injection. Plasma DPN levels declined exponentially with a half life of 0.29 hours. As expected, no DPN was detected in the plasma samples from the oil treated control animals (n = 5), and that data is not depicted on the graph. (Means ± S.E.)

Fig. 2

Performance in the light/dark box was not significantly affected by treatment with either the high (2 mg/kg) or low (0.5 mg/kg) dose of DPN. The number of light side transitions (A) and the time spent in the light side (B) were nearly identical for all groups. Similarly, behavior in the plus maze did not significantly differ between groups. The number of open arm entries (C) was not affected by either the high or low dose of DPN. Time spent in the open arms generally increased with dose (D) but the effect was not statistically significant and was largely attributable to some of the animals “freezing” on the open arms and therefore spending a disproportionate amount of time on the open arms. The proportion of time spent on the open arms and the percentage of open arm entries was also not significantly different between groups (not depicted). (Means ± S.E.)

Fig. 3

Performance in the light/dark box was not significantly affected by the 1 mg/kg dose of DPN or PPT, or the low (3 mg/kg) dose of EQ or RES. Neither the number of entries into the light side (A) nor the amount of time spent in the light side (B) differed among the groups. (Means ± S.E.)

Fig. 4

In the experiment comparing high dose (20 mg/kg) phytoestrogen exposure to DPN and PPT (1 mg/kg) exposure, DPN treated animals made fewer open arm entries relative to control animals (A) but there was no significant effect of treatment on time spent in the open arms (B). However, the DPN animals made significantly less arm entries, regardless of type, suggesting that they had lower overall levels of exploratory behavior compared to the other groups (C). Therefore, the proportion of open arm entries was analyzed (D), and no significant effect of treatment was found. These results suggest that exploratory behavior, rather than anxiety related behavior, was affected by DPN. (*P ≤ 0.05; Means ± S.E.)