The chloro-S-triazine herbicides (i.e., atrazine, simazine, cyanazine) constitute the largest group of herbicides sold in the United States. Despite their extensive usage, relatively little is known about the possible human-health effects and mechanism(s) of action of these compounds. Previous studies in our laboratory have shown that the chlorotriazines disrupt the hormonal control of ovarian cycles. Results from these studies led us to hypothesize that these herbicides disrupt endocrine function primarily through their action on the central nervous system. To evaluate this hypothesis, we examined the estrogen-induced surges of luteinizing hormone (LH) and prolactin in ovariectomized Sprague-Dawley (SD) and Long-Evans hooded (LE) rats treated with atrazine (50-300 mg/kg/day, by gavage) for 1, 3, or 21 days. One dose of atrazine (300 mg/kg) suppressed the LH and prolactin surge in ovariectomized LE, but not SD female rats. Atrazine (300 mg/kg) administered to intact LE females on the day of vaginal proestrus was without effect on ovulation but did induce a pseudopregnancy in 7 of 9 females. Three daily doses of atrazine suppressed the estrogen-induced LH and prolactin surges in ovariectomized LE females in a dose-dependent manner, but this same treatment was without effect on serum LH and prolactin in SD females. The estrogen-induced surges of both pituitary hormones were suppressed by atrazine (75-300 mg/kg/day) in a dose-dependent manner in females of both strains evaluated after 21 days of treatment. Three experiments were then performed to determine whether the brain, pituitary, or both organs were the target sites for the chlorotriazines. These included examination of the ability of (1) the pituitary lactotrophs to secrete prolactin, using hypophyosectomized females bearing pituitary autotransplants (ectopic pituitaries); (2) the synthetic gonadotropin-releasing hormone (GnRH) to induce LH secretion in females treated with high concentrations of atrazine for 3 days; and (3) atrazine (administered in vivo or in vitro) to suppress LH and prolactin secretion from pituitaries, using a flow-through perifusion procedure. In conclusion, the results of these studies demonstrate that atrazine alters LH and prolactin serum levels in the LE and SD female rats by altering the hypothalamic control of these hormones. In this regard, the LE female appeared to be more sensitive to the hormone suppressive effects of atrazine, as indicated by the decreases observed on treatment-day 3. These experiments support the hypothesis that the effect of atrazine on LH and prolactin secretion is mediated via a hypothalamic site of action.

Epidemiological, wildlife, and laboratory studies have pointed to the possible adverse health effects of chlorotriazine herbicide (i.e. , atrazine, simazine, and cyanazine) exposure. However, the cellular mechanism(s) of action of these compounds remains unknown. Recently, it was reported by Cooper et al. (2000, Toxicol. Sci. 53, 297-307) that atrazine disrupts ovarian function by altering hypothalamic catecholamine concentrations and subsequently the regulation of luteinizing hormone (LH) and prolactin (PRL) secretion by the pituitary. In this study, we examined the effect of three chlorotriazines on catecholamine metabolism in vitro using PC12 cells. Intracellular norepinephrine (NE) and dopamine (DA) concentrations and spontaneous NE release were measured following treatment with different concentrations of atrazine, simazine (0, 12. 5, 25, 50, 100, and 200 microM) and cyanazine (0, 25, 50, 100, and 400 microM) for 6, 12, 18, 24, and 48 h. Atrazine and simazine significantly decreased intracellular DA concentration in a concentration-dependent manner. Intracellular NE concentration was also significantly decreased by 100 and 200 microM atrazine and 200 microM simazine. Similarly, there was a dose-dependent inhibition of NE release with 100 and 200 microM concentrations of both compounds. Although 100 and 400 microM cyanazine increased intracellular NE concentration, 50, 100, and 400 microM cyanazine significantly increased NE release at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, but only at 400 microM. The GABA(A)-receptor agonist, muscimol (0, 0.01, 0.1, and 1.0 microM) had no effect on either the release or on intracellular catecholamine concentrations from 6 through 24 h of treatment. Cell viability was somewhat lower in the groups exposed to 100 and 200 microM atrazine and simazine. However, the reduction in viability was significant only in the highest dose of atrazine used (200 microM) at 24 h. Cyanazine did not have an effect on the viability at any of the doses tested, and the cells were functional, even up to 48 h of exposure. These data indicate that both atrazine and simazine inhibit the cellular synthesis of DA mediated by the tyrosine hydroxylase (TH), and NE mediated by dopamine beta-hydroxylase (DbetaH), and, as a result, there is a partial or significant inhibition of NE release. Cyanazine, on the other hand, stimulated the synthesis of intracellular NE, and not DA. Thus, chlorotriazine compounds presumably act at the enzymatic steps or sites of CA biosynthesis to modulate monoaminergic activity in PC12 cells.

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