The Role of Uron and Chlorobenzene Derivatives, as Potential Endocrine Disrupting Compounds, in the Secretion of ACTH and PRL

Abstract

Uron herbicides polluting the environment represent a serious concern for environmental health and may be regarded as endocrine-disrupting compounds (EDCs), which influence the regulation of human homeostasis. We aimed to investigate the effect of EDC urons (phenuron: PU, monuron: MU, and diuron: DU) and chlorobenzenes on the basal release of the adrenocorticotropic hormone (ACTH), which is a part of the adenohypophysis-adrenocortical axis. Hormone secretion in the presence of EDC was studied in two cell types: normal adenohypophysis cells (AdH) and cells of prolactinomas (PRLOMA). PRLOMA was induced in female Wistar rats by subcutaneously injecting them with estrone acetate for 6 months. AdH and PRLOMA were separated from treated and untreated experimental animals, dissociated enzymatically and mechanically in order to create monolayer cell cultures, which served as an experimental in vitro model. We investigated the effects of ED agents separately and in combination on ACTH and prolactin (PRL) release through the hypophyseal-adrenal axis. Hormone determination was carried out by the luminescent immunoassay and the radioimmunoassay methods. Our results showed that (1) uron agents separately did not change ACTH and PRL release in AdH culture; (2) ACTH secretion in arginine vasopressin- (AVP-) activated AdH cells was significantly increased by EDC treatment; (3) ED agents increased the basal hormone release (ACTH, PRL) in PRLOMA cells; and (4) EDC exposure increased ACTH release in AVP-activated PRLOMA cells. We conclude that the herbicides PU, MU, and DU carry EDC effects and show human toxicity potential.

Figure 1

Functional assay for ACTH release in normal rat adenohypophysis cultures, in vitro (n = 8–12 in each group); mean level ± SEM (pg ACTH/mg protein): AVP significantly increases (p < 0.001), whereas corticosterone alone does not alter ACTH release when compared to control; preincubation with +B inhibited AVP activation (basal: control; AVP: 8-arginine vasopressin; B: corticosterone).

Figure 2

Functional assay for ACTH release in rat prolactinoma cultures (PRLOMA), in vitro (n = 11 − 12 in each group); mean level ± SEM (pg ACTH/mg protein): compared with normal AdH cells (Figure 1). ACTH release always shows a similarly significant (p < 0.001) increase in PRLOMA cells (basal: control; AVP: 8-arginine vasopressin; B: corticosterone).

Figure 3

EDC effects in the regulation of ACTH release on normal rat AdH cultures, in vitro. Mean (ACTH level) ± SEM. The mean and SEM are calculated from n = 12. Abbreviations: B = corticosterone: 1 μg/ml; AVP = 8-arginine vasopressin: 10⁻⁶ M, +B+AVP: in combination therapy B precedes AVP administration by 20 minutes; dClB = 1,4-dichlorobenzene: 0.1 ng/ml; mClB = chlorobenzene mix: 0.1 ng/ml; PU = phenuron: 10⁻⁶ M; MU = monuron: 10⁻⁶ M; DU = diuron: 10⁻⁶ M. All EDC groups differ significantly from the control (p < 0.001) for +AVP.

Figure 4

EDC effects in the regulation of ACTH release in rat PRLOMA cultures, in vitro. Mean (ACTH level) ± SEM. The mean and SEM are calculated from n = 12. Abbreviations: B = corticosterone: 1 μg/ml; AVP = 8-arginine vasopressin: 10⁻⁶ M, +B+AVP: in combination therapy B precedes AVP by 20 minutes; dClB = 1,4-dichlorobenzene: 0.1 ng/ml; mClB = chlorobenzene mix: 0.1 ng/ml; PU = phenuron: 10⁻⁶ M; MU = monuron: 10⁻⁶ M; DU = diuron: 10⁻⁶ M. All EDC groups differ significantly from the control (p < 0.001) for basal, +AVP, and +B+AVP.

Figure 5

Effects of EDC on PRL release in normal rat AdH cultures, in vitro. Mean (PRL/prolactin/level) ± SEM. The mean and SEM are calculated from n = 12. Abbreviations: dClB = 1,4-dichlorobenzene: 0.1 ng/ml; mClB = chlorobenzene mix: 0.1 ng/ml; PU = phenuron: 10⁻⁶ M; MU = monuron: 10⁻⁶ M; DU = diuron: 10⁻⁶ M.

Figure 6

Effects of EDC on PRL release in rat PRLOMA cultures, in vitro. Mean (PRL/prolactin/level) ± SEM. The mean and SEM are calculated from n = 12. Abbreviations: dClB = 1,4-dichlorobenzene: 0.1 ng/ml; mClB = chlorobenzene mix: 0.1 ng/ml; PU = phenuron: 10⁻⁶ M; MU = monuron: 10⁻⁶ M; DU = diuron: 10⁻⁶ M. All EDC groups differ significantly from the control (p < 0.001) for basal release.