Differential Modulation of Catecholamines by Chlorotriazine Herbicides in Pheochromocytoma (PC12) Cells in Vitro

Parikshit C. Das*, William K. McElroy{dagger} and Ralph L. Cooper{dagger},1

* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; and {dagger} Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received February 16, 2000; accepted May 8, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM) and cyanazine (0, 25, 50, 100, and 400 µM) 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 µM atrazine and 200 µM simazine. Similarly, there was a dose-dependent inhibition of NE release with 100 and 200 µM concentrations of both compounds. Although 100 and 400 µM cyanazine increased intracellular NE concentration, 50, 100, and 400 µM cyanazine significantly increased NE release at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, but only at 400 µM. The GABAA-receptor agonist, muscimol (0, 0.01, 0.1, and 1.0 µM) 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 µM atrazine and simazine. However, the reduction in viability was significant only in the highest dose of atrazine used (200 µM) 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 ß-hydroxylase (DßH), 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.

Key Words: chlorotriazines; atrazine; simazine; cyanazine; PC12 cell line; dopamine; norepinephrine..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chlorotriazines were introduced as broad-spectrum herbicides in the 1950s (Eldridge et al., 1994Go; U.S. EPA, 1994). Their mode of action in plants is through an inhibition of photosynthesis by binding to a plastoquinone-binding niche on D1, a 32-kDa protein encoded by the psbA gene of the photosystem-II reaction complex, to control weeds (Worthing and Walker, 1987Go). Chronic feeding studies indicated that chlorotriazine exposure led to an early onset of mammary gland tumors in female Sprague-Dawley (SD) but not F344 rats (Thakur et al., 1998Go; Wetzel et al., 1994Go); an effect that was hypothesized to be the result of their ability to induce a premature reproductive senescence (Stevens et al., 1994Go). In this regard, atrazine exposure to the young adult Sprague-Dawley and Long-Evans-hooded female rats disrupts regular ovarian cycles (Cooper et al., 1996Go), and continued exposure to atrazine by diet in SD females will bring about an early age-related cessation of ovarian cycles (i.e., typified by a pattern of constant vaginal estrus) (Eldridge et al., 1999Go). The mode of action for these atrazine-induced changes in ovarian function is mediated by an effect on the central nervous system (CNS), which suppresses the luteinizing hormone (LH) and prolactin (PRL) surges in female rats. This conclusion is based on the following observations. First, although atrazine suppressed the LH surge in ovariectomized, estrogen-primed rat, the amount of LH released in response to exogenous gonadotropin releasing hormone (GnRH) was not modified by atrazine. Second, the pulsatile release of LH, observed in long-term ovariectomized rats not treated with estrogen, is abolished by atrazine. The LH pulses in this model are a direct result of GnRH release from the hypothalamus (Cooper et al., 1996Go). Furthermore, although atrazine suppressed the estrogen-induced secretion of PRL in females with intact pituitaries, the same treatment was without effect on serum PRL levels in hypophysectomized rats bearing ectopic pituitary autografts. That is, when the pituitary was removed from direct influence of the CNS, the effect of atrazine on the secretion of PRL was no longer present. Finally, using a pituitary perifusion system, that directly exposed the pituitary to atrazine, there was no alteration in LH or PRL secretion (Cooper et al., 2000Go).

Cooper et al., (1998) have reported that atrazine exposure (administered by gavage at 75, 150, or 300 mg/kg bw) can alter catecholamine concentrations within the rat brain. Specifically, 3 daily doses of atrazine resulted in decreased hypothalamic NE and increased hypothalamic DA concentrations. Again, these changes are consistent with the hypothesis that atrazine alters anterior pituitary hormone secretion via alterations in the brain. Numerous studies have shown that decreasing hypothalamic NE concentration will inhibit the pulsatile release of GnRH and the LH surge (Barraclough, 1992Go; Kalra and Kalra, 1983Go; Ramirez et al., 1984Go). Likewise, increasing hypothalamic DA turnover will lower PRL secretion by the pituitary (Ben-Jonathan, 1985Go). However, in these studies it was difficult to determine whether or not changes in hypothalamic catecholamine concentration occurred because the catecholamine neurons were the primary target for the chlorotriazine or because the changes in these monoamines was the result of atrazine altering other neuronal systems which, in turn, modified the activity of the catecholamine neurons. In this regard GABAergic stimulation is known to decrease catecholamine availability and regulate GnRH and LH release (Adler and Crowley, 1986Go; Akema et al., 1990Go; Fuchs et al., 1984Go; Kang et al., 1995Go; Leonhardt et al., 1995Go). Since Shafer et al., (1999) have reported that the chlorotriazines bind to a GABA receptor (GABA-R), suggest the possibility that the effect of these chemicals on catecholamine synthesis may be mediated through the GABA-R pathway.

In the present study, we examined the potential of the chlorotriazines to interfere with catecholamine synthesis and release using PC12 cells (Greene and Tischler, 1976Go). PC12 cells are neuronal cells derived from a cancerous lesion, pheochromocytoma, of the adrenal medulla of the rat and this cell line maintains the ability to synthesize both DA and NE in vitro. Catecholamine synthesis in these cells is very similar to the sympathetic neurons requiring the same metabolic enzymes, and are regulated by similar second messenger systems in mammals (Greene and Tischler, 1976Go). These cells provide an alternative model to examine whether or not atrazine, or two other triazines (simazine and cyanazine) alter the constitutive synthesis of dopamine and norepinephrine.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Dulbecco's modified essential medium (D-MEM) with L-glutamine and high glucose and D-MEM/F-12 medium, Fetal bovine serum (FBS), horse serum (HS), 1 M Hepes buffer, 7.5% (w/v) NaHCO3 solution, and other tissue culture-related products were purchased from Gibco BRL, Life Technologies, Grand Island, N.Y. Rat tail collagen type VII, bovine-serum albumin (BSA) initial fraction, L-ascorbic acid, and other chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Tissue culture flasks, cell culture clusters, and all other tissue culture-related plastic wares were purchased from Corning Costar Corp., Cambridge, MA. The chlorotriazines: atrazine [2-chloro-4-(ethylamino)-6-) isopropylamino)-s-triazine], simazine [2-chloro-4,6-bis(ethylamino)-s-triazine], and cyanazine [2-((4-chloro-6-(ethylamino)-s-triazine-2-yl)amino)-2-methylpropionitrile] were generously provided by Novartis Crop Protection, Inc., Greensboro, NC. The purity of atrazine, simazine, and cyanazine was 97.1%, 98.3%, and 97.5%, respectively. Although, the identities of the 2–3% impurities in these chlorotriazines are not known. HPLC grade water previously filtered and deionized through a milli-Q system was used to make all buffers. All three chlorotriazines, atrazine, simazine, and cyanazine were dissolved in 100% ethyl alcohol as stock solutions at a 30 mM concentration. The respective concentration of ethyl alcohol (maximum 6.7 µl/ml of media) was added into each corresponding control in all experiments.

Cell culture and treatment.
PC12 cells, originally described in detail by Greene and Tischler (1976) and generously provided by Dr. Tim Shafer (Neurotoxicology Division, NHEERL, U.S. EPA, RTP, NC) were grown in 75 cm2, 0.2 mm vented-cap tissue culture flask containing D-MEM, 4, 500 mg/l D-glucose, 7.5% FBS, 7.5% HS, 2 mM L-glutamine, 2 mM Hepes buffer and 44 mM NaHCO3 at 37°C under an atmosphere of 5% CO2 plus 95% air. The culture medium was replenished at 4-day intervals, based on the doubling time of PC12 cells (Greene and Tischler, 1976Go). Our lab has found this cell line quite useful for evaluating the effect of selected compounds on catecholamine synthesis including those known to stimulate DA and NE such as 8bromo-cAMP, dexamethasone, forskolin, as well as those known to inhibit DA and NE synthesis such as dimethyldithiocarbamate, {alpha}-methyl-p-tyrosine, and fusaric acid. All experiments were performed by using the cells in given passage numbers 9 to 11 to keep inter-experimental variability at a minimum. In spite of that, the control values did vary between the experiments (with lower control values being observed in the cyanazine experiments). This may be due to the inherent variability of intracellular catecholamines in this cell line (Greene and Tischler, 1976Go) and the use of different batches of cells for these experiments. The number of viable/live cells in the suspension were counted by trypan blue (0.4% in PBS) exclusion in a hemocytometer and routinely contained 97–98% viable/live cells at the time of each experiment. Cells were plated in 24-well rat collagen pre-coated cell culture plate, containing 0.5 ml complete D-MEM culture medium in each well, at a density of 2.5 x 105 cells/well, and incubated for 2 h. Then the D-MEM culture medium was replaced with D-MEM/F-12 medium supplemented with 2 mM L-glutamine, 2 mM Hepes buffer, 2.5 g/l BSA, 44 mM NaHCO3 solution, and 1 mM L-ascorbic acid (added immediately before use), and incubated for ~12 h. The medium was then replaced with either fresh complete D-MEM/F-12 medium plus vehicle or different concentrations of atrazine, simazine, cyanazine, or muscimol. Cells were incubated for the specified time periods as indicated.

PC12 cells were exposed to atrazine, simazine (0.0., 12.5, 25.0, 50.0, 100.0, and 200.0 µM), cyanazine (0.0, 25.0, 50.0, 100.0, and 400.0 µM) and muscimol (0.0, 0.01, 0.1, and 1.0 µM). The concentrations of DA and NE released in the medium and intracellular DA and NE were measured in the PC12 cells exposed to atrazine and simazine at 6, 12, 18, and 24 h, and cyanazine at 6, 12, 18, 24, 36 and 48 h. The concentrations of DA and NE released in the medium at 6, 12, and 24 h, and in the cell at 6, 12, 18, 24, and 36 h were measured following muscimol treatment.

Quantification of dopamine and norepinephrine.
Following the specified treatment period, both medium and cells devoid of medium were harvested in chilled HPLC mobile-phase buffer containing 4–8% acetonitrile, 115 mM Na2HPO4, 0.19 mM EDTA, 3 mM 1-heptanesulfonic acid sodium salt in HPLC-grade water, previously filtered and deionized through a Milli-Q system (Millipore, MA), and stored at –20°C until prepared for DA and NE assays. At each of the specified time points, the concentrations of DA and NE released into the medium, and also in the cell suspension (i.e., intracellular contents) were determined by HPLC and electrochemical detection, as described previously (Goldman et al., 1994Go) using a mobile-phase buffer containing 4–8% acetonitrile. Prior to the assay, the medium was diluted in HPLC mobile-phase buffer and centrifuged at 13,000 rpm for 3 min. To determine intracellular DA and NE, the cells were sonicated briefly (10 s) using a Fisher Model 300 sonic dismembrator and centrifuged at 13,000 rpm at 4°C for 15 min. The supernatants were used for subsequent DA and NE assay. The concentration of DA in the medium was undetectable.

Cell viability and functional status assay following chlorotriazine treatment.
Following treatment with different concentrations of the 3 chlorotriazines for the specified time periods, cells were harvested to be evaluated for viability as a cell suspension in isotonic PBS. Trypan blue exclusion assay was used to determine the viability of cells, using a hemocytometer. The viability of cells was expressed as the % change and data are summarized from a representative experiment in Figure 1Go. Cell viability did appear to be affected at the doses of atrazine and simiazine tested, but not after exposure to cyanazine. However, the decrease observed was only statistically significant at the 200 µM concentration of atrazine at 24 h. It is unlikely that this small change in cell viability observed after atrazine or simazine exposure contributed to the changes in catecholamine concentrations to any significant extent. Thus, although the higher doses of atrazine and simazine may have compromised the cells` viability, the suppression of DA and NE by these 2 compounds occurred at substantially lower doses.



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FIG. 1. Time course of the number of viable cells (% change) following treatment with atrazine, simazine and cyanazine. PC12 cell cultures were exposed to atrazine and simazine respectively with 0 µM (white pattern bar), 50 µM (1st/left-hatched pattern bar), 100 µM (2nd./right-hatched pattern bar), and 200 µM (square pattern bar) concentrations for the indicated time points. Similarly, PC12 cell cultures were treated with 0 µM (white pattern bar), 50 µM (1st/left hatched pattern bar), 100 µM (2nd/right hatched pattern bar), and 400 µM (square pattern bar) cyanazine concentrations for the indicated time points. Each panel represents the number of viable cells (% change) following treatment. Each bar is the mean of % change value of 6 cultures ± SEM, representing one experiment only. An asterisk indicates the level of significance, p < 0.05.

 
In order to determine whether the depletion of nutrients in the medium, accumulation of toxic by-products of metabolism, and change in medium composition influenced the ability of PC12 cells to synthesize DA and NE following exposure to cyanazine for 48 h, the cells were exposed to cyanazine at different concentrations (0–400 µM) each in quadruplicate wells in duplicate sets in a representative experiment. In one set, cells were exposed to cyanazine up to 24, 36, and 48 h; while in another set, the culture medium was harvested at 18 h and replaced with fresh medium plus vehicle or cyanazine in corresponding wells in all groups, then incubated for up to 24, 36, and 48 h. Following complete exposure, the medium and the cells were harvested at the specified time points for the determination of DA and NE similarly as indicated under the Analysis of DA and NE section. Data from both sets were compared between the corresponding treatment group at different time points. Data indicated a sustained concentration of released NE in the medium and a sustained intracellular DA and NE level in the medium replaced group, as opposed to a slight decrease in both released NE and intracellular DA and NE levels in the continuously exposed group of cells (data not shown). The results indicate that a slight decrease in catecholamine levels at 36 and 48 h in the continuously exposed group was probably due to impaired precursor molecules in the medium and the accumulation of toxic by-products of metabolism; while in the medium replaced set, cyanazine exposure for up to 48 h did not affect the functional status of the cells, as indicated by both released NE and intracellular DA and NE levels.

Statistical analysis.
Data are expressed as mean ± SEM and analyzed by ANOVA (GraphPad InStat, GraphPad Software, San Diego, CA). The Bonferroni Multiple Comparison Test was performed to evaluate the level of significance between control and the specific treatment groups. The level of significance for all statistical tests was set at {alpha} = 0.05. It is important to note that all comparisons were made with concomitant run controls so that such inter-assay variance would not confound the finding results.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Time- and Concentration-Response Effects of Atrazine and Simazine on the Release of Norepinephrine and Intracellular Contents of Dopamine and Norepinephrine
The intracellular concentration of DA and NE and the concentration of NE in the medium following exposure to atrazine and simazine are shown in Figures 2Go (atrazine) and 3Go (simazine). In each case, there was a concentration-dependent decrease in the catecholamine concentration. However, this was most evident in the decline in intracellular DA, where there was a significant depletion observed at 12.5 µM for atrazine and 50 µM for simazine at each of the time points. The concentration of NE in these cells was reduced by exposure to 100 µM atrazine at 12 through 24 h; a similar suppression was observed only after exposure to 200 µM simazine at all time points examined. Norepinephrine release was also suppressed by both chlorotriazines. Atrazine decreased the amount of NE released into the medium, with concentrations of 50–200 µM while 200 µM of simazine was required to significantly lower the release of norepinephrine.



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FIG. 2. Time course of released NE and intracellular DA and NE following exposure to atrazine. PC12 cell cultures were exposed to atrazine with 0 µM (white pattern bar), 12.5 µM (1st/left-hatched pattern bar), 25 µM (2nd/right-hatched pattern bar), 50 µM (square pattern bar), 100 µM (horizontal pattern bar), and 200 µM (vertical pattern bar) concentrations for the indicated time points. Panel A represents the amount of NE released into the medium, while panels B and C represent the intracellular DA and NE concentrations respectively. Each bar is the value of the mean ± SEM of >= 10 cultures. Data were duplicated in subsequent experiment. An asterisk indicates the significance level (p < 0.05) at each time point, as compared to the respective control.

 


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FIG. 3. Time course of released NE and intracellular DA and NE following exposure to simazine. PC12 cell cultures were exposed to simazine with 0 µM (white pattern bar), 12.5 µM (1st/left-hatched pattern bar), 25 µM (2nd/right-hatched pattern bar), 50 µM (square pattern bar), 100 µM (horizontal pattern bar), and 200 µM (vertical pattern bar) concentrations for the indicated time points. Panel A represents the amount of NE released into the medium, while panels B and C represent the intracellular DA and NE concentrations respectively. Each bar is the value of mean ± SEM of >= 10 cultures. Data were duplicated in the subsequent experiment. An asterisk indicates the significance level (p < 0.05) at each time point as compared to the respective control.

 
Time- and Concentration- Response Effect of Cyanazine on the Norepinephrine Release and Intracellular Dopamine and Norepinephrine Content
Exposure to cyanazine resulted in a different effect on intracellular DA and NE and release of NE as compared with atrazine and simazine. Cyanazine increased intracellular NE (25 µM and greater at 18 h). A similar increase in intracellular NE was observed at 24 through 48 h at both 100 and 400 µM cyanazine. Norepinephrine release in these cells was enhanced by cyanazine at 24 h (25–400 µM), 36 (50–400 µM) and 48 h only at the 100 µM concentration. A 4-fold increase in cyanazine concentration (400 µM) was required to cause a reduction in intracellular DA as compared with atrazine and simazine (Fig. 4Go).



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FIG. 4. Time course of released NE and intracellular DA and NE following exposure to cyanazine. PC12 cell cultures were exposed to cyanazine with 0 µM (white pattern bar), 25 µM (1st/left-hatched pattern bar), 50 µM (2nd/right-hatched pattern bar), 100 µM (square pattern bar), and 400 µM (horizontal pattern bar) concentrations for the indicated time points. Panel A represents the amount of NE released into the medium, while panels B and C represent the intracellular DA and NE concentrations, respectively. Each bar is the value of the mean ± SEM of >= 8 cultures. Data were duplicated in the subsequent experiment. An asterisk indicates the significance level (p < 0.05) at each time point as compared to the respective control.

 
In order to verify whether muscimol binding site of GABA-R is involved in the chlorotriazine-induced alteration of catecholamine, exposure of PC12 cells to the GABA-R agonist, muscimol was without effect on both intracellular catecholamine concentrations and the release of norepinephrine (see Fig. 5Go).



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FIG. 5. Time course of released NE and intracellular DA and NE following exposure to muscimol. PC12 cell cultures were exposed to muscimol with 0 µM (white pattern bar), 0.01 µM (1st/left- hatched pattern bar), 0.1µM (2nd/right-hatched pattern bar), 1.0 µM (square pattern bar) concentrations for the indicated time points. Panel A represents the amount of NE released into the medium, while panels B and C represent the intracellular DA and NE concentrations respectively. Each bar is the value of the mean ± SEM of 6 cultures, where experiments were duplicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data reported here demonstrate that exposing PC12 cells to chlorotriazine herbicides alters catecholamine synthesis. Atrazine and simazine led to a significant decrease in intracellular DA and NE concentrations and NE release into the medium. In contrast, the same exposure to cyanazine resulted in a significant increase in intracellular NE concentration and release. Importantly, it should be noted that catecholamine synthesis and release was affected at doses well below where any change in cell viability was found. The ability of atrazine to suppress NE synthesis and release in PC12 cells is in agreement with our preliminary in vivo data in which we found a suppression of hypothalamic NE concentration following 3 daily doses of atrazine (Cooper et al., 1998Go). However, these same doses of atrazine led to an increase in hypothalamic DA concentration in the in vivo studies. Thus, there is a fundamental difference in the effect of atrazine on DA metabolism when the in vitro and in vivo results are compared. Although it is difficult to relate in vitro observations to in vivo responses, similar discrepancies have been reported following treatment with other well-known inhibitors of NE synthesis. For example, the dithiocarbamates [diethyldithiocarbamate (DEDC), disulfiram, metam sodium, and the common metabolite dimethyldithiocarbamate (DMDC)] (Goldman et al., 1994Go; Goldstein and Nakaijima, 1967Go; Hashimoto et al., 1965Go; Szmigielski, 1975Go) have been shown to alter catecholamine. These compounds decrease CNS NE by chelation of the copper-containing site of dopamine-ß-hydroxylase (DßH), the enzyme responsible for the synthesis of NE from the precursor DA. In addition, Goldman et al. (1994) have reported that in vivo exposure to sodium N-methyldithiocarbamate (SMD), also known as metam sodium or its bioactive metabolite methylisothiocyanate (MITC), is associated with a concomitant increase in hypothalamic DA and decrease in NE concentrations (Goldman et al., 1994Go). However, when examined in vitro using PC12 cells, these same compounds, DEDC and DMDC, were found to decrease DA (Montine et al., 1995Go). We have observed a similar decrease in intracellular DA and NE in PC12 cells exposed to DMDC (Das, unpublished observation). Thus, both the in vivo and in vitro profile of the atrazine and simazine in the present study are remarkably similar to the dithiocarbamates. Since we did not examine the effect of cyanazine on hypothalamic catecholamine concentrations, and we are unaware of any other study in which the catecholamines have been measured following cyanazine exposure in vivo (or in vitro), we cannot conclude that there is a similar in vivo/ in vitro relationship for this triazine.

The specific cellular mechanism through which the atrazine and simazine interfere with catecholamine synthesis was not determined. Since the changes observed following in vivo and in vitro exposure to atrazine and simazine are similar to those observed following exposure to the dithiocarbamates, it is tempting to speculate that they act through a common mechanism (i.e., blocking the enzymes that are involved in synthesis). However, there are fundamental differences in the action of the chlorotriazines and dithiocarbamates that would imply that the mode of action of these chemicals might be different. Most notable is the difference in the time course for the depletion of the catecholamines. The dithiocarbamates result in a more rapid (in terms of time) depletion of DA and NE than that seen for atrazine and simazine. This raises the possibility that the modification of tyrosine hydroxylase (TH), as indicated by a decrease in intracellular DA, or in DßH activity, as indicated by the increase in intracellular NE, was the result of an effect of the herbicides on the intracellular regulation of TH and DßH. The activity of both enzymes have been shown to be regulated through various membrane-mediated second messenger signal transduction mechanisms, e.g., calcium ions (Ca++) and protein kinase C (PKC). Protein kinase A (PKA) also plays an important role in basal, as well as Ca++ and cyclic AMP (cAMP)-inducible expression of those enzymes involved in catecholamine synthesis in PC12 cells and in bovine adrenal medullary cells (Hwang et al., 1997Go; Kim et al., 1993Go). This effect on gene expression is mediated through the phosphorylation of cAMP response element binding protein (CREB) (Dash et al., 1991Go; Sheng et al., 1991Go). Dexamethasone (synthetic form of glucocorticoid) can also alter mRNA levels of TH and DßH through transcriptional activation in pheochromocytoma cultures within 24 h (Hwang and Joh, 1993Go; Kim et al., 1993Go, 1994Go; Lewis et al., 1987Go). However, more time (between 48 to 72 h) is required before an increase in the specific activity of these enzymes reaches maximal levels (e.g., 3.6- to 8.0-fold greater than non-stimulated cells) (Kim et al., 1993Go; Tank et al., 1986Go). The resulting changes in enzymatic activity leads to changes in intracellular catecholamine concentrations. Thus, there may be a correlation between the reported duration of altered TH or DßH activity and the duration of altered concentration of intracellular DA and NE by atrazine, simazine or cyanazine. However, the specific mechanism(s) is not yet known.

In vivo, activation of the GABA-R within the hypothalamus and anterior pituitary has been shown to modify GnRH and LH release respectively (Adler and Crowley, 1986Go; Akema et al., 1990Go; Fuchs et al., 1984Go; Kang et al., 1995Go; Leonhardt et al., 1995Go). PC12 cells possess GABAA-R Cl ion channels, and the GABAA-R subunits identified in PC12 cells include ß3, {gamma}2L, {gamma}2s, and {delta} subunits (Tyndale et al., 1994Go). These cells also contain the benzodiazepine-binding site (Miller et al., 1988Go; Morgan et al., 1985Go). Interestingly, these cells do not respond to GABA-ergic stimulation (Hales and Tyndale, 1994Go; Wisden, Moss, 1997Go) and in this study, the GABA-ergic agonist muscimol was without effect. Using rat brain cortical synaptoneurosomes, Shafer et al., (1999) reported that cyanazine disrupts benzodiazepine, but not muscimol or TBPS (Cl channel) binding. Interestingly, these authors also report that cyanazine binding occurred at a lower concentration than atrazine binding. Simazine binding was not examined in our study. Thus, atrazine- and simazine-mediated inhibition and cyanazine-induced stimulation of catecholamine in PC12 cells may be mediated via GABAA-R, independent of muscimol binding-site pathway.

The primary purpose of these experiments was to determine whether or not a change in CA biosynthesis would occur following chlorotriazine exposure using PC12 cells, and whether or not the direction of change would be similar to that observed in vivo. The concentrations of the chlorotriazines used in this series of experiment range from 2.69–43.14 ppm for atrazine, 2.52–40.34 ppm for simazine, and 6.01–48.14 ppm for cyanazine. Although it is difficult to compare in vitro and in vivo results, the concentrations used in this study do appear to fall within those levels observed following in vivo exposure. For example, adverse effects following in vivo exposure have been noted for mammary tumors with LOAELs of 70–400 ppm (Stevens et al., 1994Go). Pubertal exposure to atrazine was found to delay preputial separation (LOAEL = 12.5 mg/kg or ~175 ppm) (Stoker et al., 2000Go). A significant increase in prostatitis in the male offspring occurred following atrazine treatment (LOAEL = 25 mg/kg or ~350 ppm) to the dam during the initial phase of lactation (Stoker et al., 1999Go). Atrazine also interferes with the estrogen-induced increase of radiolabeled thymidine incorporation in the uterus of an atrazine-treated rat (LOEL = 30 mg/kg or ~420 ppm) (Tennant et al., 1994Go). The order of potency for these in vivo effects in the female rat following oral exposure is cyanazine > atrazine >= simazine (Eldridge et al., 1994Go; Cooper et al., 2000Go).

In summary, the effects of atrazine and simazine on catecholamine synthesis in PC12 cells observed in the present study support the hypothesis that alterations in CNS NE and DA in vivo underlie the changes observed in pituitary hormone secretion following in vivo exposure to atrazine. It is well known that the ovulatory surge of LH in the female rat is under hypothalamic, particularly noradrenergic control, and that compounds that interfere with NE synthesis will inhibit the pulsatile release of GnRH, LH secretion and the ovulatory surge of LH (Barraclough, 1992Go; Cooper et al., 1999Go). Whether a similar series of events underlies the effect of cyanazine on reproductive function, in vivo, remains to be determined. Further studies evaluating the triazine-induced changes in catecholamine synthesis to determine whether or not these effects are mediated through a direct effect on the synthetic enzymes or the second-messenger systems involved in their regulation are in progress.


    ACKNOWLEDGMENTS
 
The authors express their thanks to Dr. Tim J. Shafer, Neurotoxicology Division, and Dr. Susan C. Laws, Endocrinology Branch, Reproductive Toxicology Division, NHEERL, U.S. EPA, Research Triangle Park, NC for commenting on this manuscript.


    NOTES
 
This study was presented in part as a poster exhibit at the 38th Annual Meeting of the Society of Toxicology, New Orleans, Louisiana, 1999.

This manuscript has been reviewed following the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at the Endocrinology Branch, MD-72, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-5138. E-mail: cooper.ralph{at}epamail.epa.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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