Alteration of Catecholamines in Pheochromocytoma (PC12) Cells in Vitro by the Metabolites of Chlorotriazine Herbicide

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, MD-72, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received August 1, 2000; accepted September 20, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of four major chlorotriazine metabolites on the constitutive synthesis of the catecholamines dopamine (DA) and norepinephrine (NE) were examined, using undifferentiated PC12 cells. NE release and intracellular DA and NE concentrations were quantified, for up to 24 h after initiation of treatment with different concentrations, ranging from 0 to 400 µM, of the metabolites hydroxyatrazine (HA), 2-amino-4-chloro-6-isopropylamino-s-triazine (deethylchlorotriazine), 2-amino-4-chloro-6-ethylamino-s-triazine (deisopropylchlorotriazine), and diaminochlorotriazine (DACT). Hydroxyatrazine significantly decreased intracellular DA and NE concentrations in a dose- and time-dependent manner. This metabolite also caused a significant inhibition of NE release from the cells. In contrast, deethylchlorotriazine and deisopropylchlorotriazine significantly increased intracellular DA concentration following exposure to 50–200 µM from 12 to 24 h. Intracellular NE was significantly reduced at these same concentrations of deethylchlorotriazine at 24 h while the concentration of NE in PC12 cells exposed to deisopropylchlorotriazine was not altered at any dosage or time point measured. NE release was decreased at 18 (200 µM) and 24 h (100 and 200 µM) following exposure to deethylchlorotriazine and at 24 h (100 and 200 µM) following deisopropylchlorotriazine. DACT, at the highest concentration (160 µM), significantly increased intracellular DA and NE concentrations at 18 and 24 h. NE release was also increased at 40–160 µM at 24 h. The viability of the PC12 cells was tested using the trypan blue exclusion method. Following 18 to 24 h of treatments with HA, cell viability was reduced 10–12% at the two higher concentrations (200 and 400 µM), but, with other metabolites, the viability was reduced by only 2 to 5% at the highest concentrations. These data indicate that HA affects catecholamine synthesis and release in PC12 cells in a manner that is similar to synthesis of atrazine and simazine. On the other hand, deethylchlorotriazine and deisopropylchlorotriazine altered catecholamine synthesis in a manner similar to that observed in the rat brain following in vivo exposure (i.e., increased DA and decreased NE concentration), whereas DACT appeared to produce an increase in NE release as well as in the intracellular DA and NE concentrations. Overall, these findings suggest that the catecholamine neurons may be a target for the chlorotriazines and/or their metabolites, that the metabolites produce a unique pattern of catecholamine response, and that all of the changes were seen within the same range of doses.

Key Words: chlorotriazine metabolites; hydroxyatrazine; deethylchlorotriazine; deisopropylchlorotriazine; diaminochlorotriazine; PC12 cells; dopamine; norepinephrine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorotriazine herbicides have been widely used in the United States and other countries worldwide for about 40 years (Eldridge et al. 1994Go; U.S. EPA, 1994Go). Specifically, these compounds block photosynthesis by inhibiting the function of the psbA gene (Worthing and Walker, 1987Go). The environmental fate of atrazine has been investigated extensively over the last 4 decades, and its retention and transport in soil have been reviewed in recent years (Flury, 1996Go; Koskinen and Clay, 1997Go; Ma and Selim, 1996Go). Atrazine is persistent in the soil (Seiler et al., 1992Go), and has been detected in ground and surface waters at concentrations exceeding the Environmental Protection Agency's maximum contaminant level of 3 ppb (Kello, 1989Go).

The metabolites identified in the environment and in the urine of mammals (including humans) are presented in Figure 1Go. In the environment, one of the major atrazine degradation pathways is chemical hydrolysis to 6-hydroxyatrazine in the surface soil (Harris, 1967Go; Muir and Baker, 1978Go), something that is favored by low pH (5.5–6.5), elevated moisture content, high temperature, and increased organic matter levels (Ahrens, 1994Go; Koskinen and Clay, 1997Go; Ma and Selim, 1996Go). Microbial degradation also takes place in the soil. This process yields 6-chloro-N-[1-methylethyl]-1,3,5-triazine-2,4-diamine and 6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine. In addition, atrazine is also degraded by microbial enzymes to 6-hydroxyatrazine (De Souza et al., 1998Go; Mandelbaum et al., 1993Go). Photodegradation of atrazine can also occur in the soil. This process also produces deethylated atrazine (deethylchlorotriazine) and N-deethyl-N-demethylethyl atrazine (i.e., diaminochlorotriazine) (Ahrens, 1994Go; Koskinen and Clay, 1997Go; Ma and Selim, 1996Go). The microbial degradation of chlorinated products of atrazine has higher mobility and greater potential to contaminate groundwater than hydroxyatrazine (Sorenson et al., 1993Go), which is strongly adsorbed onto surface soils (Ma and Selim, 1996Go). The half-lives for deethylatrazine (26 days), deisopropylchlorotriazine (17 days) and hydroxyatrazine (121 days) have been documented in soil samples from western Tennessee (Winkelmann and Klaine, 1991Go), whereas in a sandy-till aquifer, the half-lives of these metabolites is considerably longer, ranging from 2700 to 3400 days (Levy and Chesters, 1995Go). No significant degradation of atrazine occurred after 270 days in aquifer slurries in Wisconsin, indicating that atrazine itself is resistant to degradation in groundwater and aquifer sediments (Rodriguez and Harkin, 1997Go), and persists for a long time in the environment, potentially affecting human and wildlife populations.



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 1. Chlorotriazine degradation occurs mainly via environmental routes and through mammalian systems, as supported by the in vitro study. The major metabolites that are identified in the urine of mammals and human are indicated in the depicted scheme. References: (a) Koskinen and Clay, 1997; (b) Lamoureux et al., 1998; (c) Bakke et al., 1972; Bradway and Moseman, 1982; (d) Erickson et al., 1979; (e) Catenacci et al., 1990; Ikonen et al., 1988; (f) Adams et al., 1990.

 
The metabolism of the chlorotriazines by different species has also been studied. In vitro studies, using livers of rat, mouse, goat, sheep, pig, rabbit and chicken, revealed that atrazine is metabolized in two phases. First, atrazine is catalyzed by cytochrome P450 to form the monodealkylated metabolites of atrazine (Adams et al., 1990Go; Hanioka et al., 1998Go). These metabolites are then conjugated with glutathione at the 2 position (Gudderwar and Dauterman, 1979Go). Atrazine itself can also be partly conjugated with glutathione at the 2 position (Adams et al., 1990Go). The chemical structures of chlorotriazines and their major metabolites are presented in Figure 2Go. In the female rat, four types of urinary metabolites have been reported, including 2-hydroxyatrazine, two monodealkylated analogs [2-chloro-4-amino-6-(ethylamino)-s-triazine, 2-chloro-4-amino-6-(isopropylamino)-s-triazine], and ammeline (Bakke et al., 1972Go). Diaminochlorotriazine was also identified in the blood of adult male rats (Bradway and Moseman, 1982Go). In swine, the primary urinary metabolite is deethylchlorotriazine (Erickson et al., 1979Go), whereas in occupationally exposed male humans, the two monodealkylated analogs (deethylchlorotriazine and deisoproplychlorotriazine) were present in urine (Catenacci et al., 1990Go; Ikonen et al., 1988Go).



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 2. Chemical structures of chlorotriazine herbicides, and the major metabolites. Atrazine, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine; simazine, 2-chloro-4,6-bis(ethylamino)-s-triazine; deisopropyl-s-chlorotriazine, 2-chloro-4-ethylamino-6-amino-s-triazine; deethyl-s-chlorotriazine, 2-chloro-4-amino-6-isopropylamino-s-triazine; diamino-s-chlorotriazine, 2-chloro-4,6-diamino-s-triazine (Budavari, 1996Go).

 
Chlorotriazines and their metabolites have been reported to alter physiological processes. Chronic feeding studies revealed that dietary atrazine exposure (at 75 and 400 ppm in food) of Sprague-Dawley female rats caused premature reproductive senescence and an earlier onset of mammary gland tumors (Eldridge et al., 1994Go; Pinter et al., 1990Go; Stevens et al., 1994Go; Thakur et al., 1992Go; Wetzel et al., 1994Go). Similar treatment in Fischer-344 females was without effect. It was hypothesized that this difference in response was the consequence of the different pattern of reproductive aging that is normally present in these two strains. Other studies have shown that atrazine can disrupt normal ovarian cycling in young adult female rats (Cooper et al., 1996Go; Eldridge et al., 1994Go, 1999Go). Cooper and colleagues have examined the mechanisms responsible for the alterations of ovarian cycling (Cooper et al., 1996Go, 1999Go, 2000aGo) and found that atrazine causes a dose-dependent decrease in the ovulatory surge of luteinizing hormone (LH) and a concomitant suppression of prolactin secretion from the pituitaries of Sprague-Dawley and Long-Evans hooded rats (Cooper et al., 2000). Atrazine was also found to cause a dose-dependent inhibition of suckling-induced prolactin release in the lactating female Wistar rat (Stoker et al., 1999Go). Several experiments have been conducted to demonstrate that the primary site of atrazine action is within the hypothalamus (Cooper et al., 2000aGo). It can cause a decrease in hypothalamic norepinephrine (NE) and an increase in hypothalamic dopamine (DA) concentrations (Cooper et al., 1998Go), effects that are consistent with a decrease in LH and prolactin secretion, respectively. Numerous studies have shown that decreasing hypothalamic NE concentration will inhibit the pulsatile release of gonadotropin releasing hormone (GnRH) and the LH surge (Barraclough, 1992Go; Ramirez et al., 1984Go; Kalra and Kalra, 1983Go) and that DA inhibits prolactin secretion (Ben-Jonathan, 1985Go). The possibility that these catecholaminergic neurons are direct targets for atrazine and other chlorotriazines was further suggested in a recent study using the sympathetic neuronal PC12 cell line (Das et al., 2000Go). Atrazine, simazine and cyanazine were shown to modulate the constitutive synthesis of DA and NE suggesting that the atrazine-induced reduction in hypothalamic NE could well be the result of the direct effect of atrazine or its metabolites on the CNS cells.

In the present study, we used undifferentiated PC12 cells to examine the effect of four primary metabolites of atrazine on catecholamine metabolism in an attempt to determine whether any of these compounds would modify catecholamine synthesis and release, to characterize the pattern of change present, and to establish the relative potencies for such effects. The metabolites selected are shown in Figure 2Go. Recent studies in our laboratory indicate that several of these metabolites, administered in vivo, affect ovarian cyclicity (Cooper et al., 2000bGo), producing effects consistent with those of atrazine. PC12 cells have been well characterized and are very similar to the sympathetic neurons in mammals, and provide an in vitro alternative testing model (Greene and Tischler, 1976Go, 1982Go) to examine whether the major chlorotriazine metabolites alter the constitutive synthesis of dopamine and norepinephrine.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals/reagents.
Specific chemicals/reagents include 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, NY. Rat tail collagen type VII, bovine serum albumin (BSA) initial fraction, and L-ascorbic acid, were obtained 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. Hydroxyatrazine (HA), 2-amino-4-chloro-6-isopropylamino-s-triazine (deethylchlorotriazine), 2-amino-4-chloro-6-ethylamino-s-triazine (deisopropylchlorotriazine), and diaminochlorotriazine (DACT) were generously provided by Novartis Crop Protection, Inc., Greensboro, NC, and their purity was 97.1%, 98.2%, 94.5%, and 95.7% respectively. The composition of about 2–5% of impurities in these compounds is not known. HPLC-grade water previously filtered and deionized through a Milli-Q Water System, Millipore Corporation, Bedford, MA, was used to prepare all buffers. All 4 chlorotriazine metabolites, HA, deethylchlorotriazine, deisopropylchlorotriazine, and DACT were dissolved in 100% ethyl alcohol as stock solutions at 15–30 mM concentration and used after a serial dilution in culture medium. A comparable concentration of ethyl alcohol (maximum 26.8 µl/ml of media or less) was added into each corresponding control in all experiments.

PC12 cell culture.
PC12 cells were originally generated and described in detail by Greene and Tischler (1976) and generously provided by Dr. Tim Shafer (Neurotoxicology Division, NHEERL, U.S. EPA, RTP, NC). They were grown in 75 cm2, 0.2 mm vented cap tissue culture flasks containing D-MEM; 4,500 mg/l D-glucose, 7.5% fetal bovine serum (FBS), 7.5% horse serum (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). Previous experiments in our lab have shown that the catecholamine synthesis in the PC12 cells can be stimulated or inhibited by a number of compounds (Das et al., 2000Go). All experiments were performed by using the same stock of cells in a passage number of 9-to-11, to keep inter-experimental variability at a minimum. 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 different concentrations of vehicle or HA, deethylchlorotriazine, deisopropylchlorotriazine, or DACT along with a standard concentration of a stimulatory (e.g., forskolin) or inhibitory (e.g., fusaric acid) compound (as positive or negative internal controls). Cells were incubated for the specified time periods as indicated.

Quantification of Dopamine and Norepinephrine
Treatment of cells.
A similar treatment paradigm was followed with the metabolites as reported earlier for atrazine, simazine and cyanazine (Das et al., 2000Go). Briefly, PC12 cells were exposed to HA (0.0., 6.25, 12.5, 25.0, 50.0, 100.0, 200.0, or 400.0 µM) for 3, 6, 12, 18, or 24 h; deethylchlorotriazine and deisopropylchlorotriazine (0.0, 12.5, 25.0, 50.0, 100.0, or 200.0 µM) and DACT (0, 10, 20, 40, 80, or 160 µM), for 6, 12, 18, or 24 h. These concentrations are equivalent to 1.23–78.88 ppm for HA, 2.34–37.4 ppm for deethylchlorotriazine, 2.16–34.6 ppm for deisopropylchlorotriazine, and 1.45–55.29 ppm for DACT. These values were selected for comparison with concentrations of parent atrazine, simazine, and cyanazine shown to inhibit or stimulate the constitutive synthesis and release of catecholamines in PC12 cells (Das et al., 2000Go). The experiments were duplicated.

Collection of culture medium and cells.
Following the specified treatment period, both culture medium and cells devoid of medium, were harvested in chilled HPLC mobile-phase buffer containing 4% 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 Water System, and stored immediately at –20°C until assayed for DA and NE by HPLC.

Cell Viability Following Treatment
Trypan blue exclusion essay.
Prior to examining changes in catecholamine release or intracellular concentration, the viability of the cells exposed to the different concentrations of the metabolites was examined. Following treatment with different concentrations of the 4 chlorotriazine metabolites for the specified time periods (i.e., HA [0, 100.0, 200.0, and 400.0 µM], deethylchlorotriazine and deisopropylchlorotriazine [0, 50.0, 100.0, and 200.0 µM], and DACT [0, 40, 80, and 160 µM], for 6, 18, and 24 h), the cells were harvested to be evaluated for viability as cell suspensions in isotonic PBS solution. Trypan blue exclusion assay was used to determine viability of cells using a hemocytometer. The numbers of viable/live cells in 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.

Quantification of DA and NE concentrations by HPLC with ECD.
At each of the specified time points following exposure, the concentrations of DA and NE released into the medium and in the cell suspensions (i.e., intracellular contents) were determined by HPLC and electrochemical detection (ECD) as described previously (Goldman et al., 1994Go) using a mobile phase buffer containing 4% acetonitrile. Prior to the assay the medium was diluted in HPLC mobile phase buffer and centrifuged at 13,000 rpm for 3 min. to remove suspended cells or any cell debris. To determine intracellular DA and NE, the cells in HPLC mobile-phase buffer 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 further diluted in HPLC mobile phase buffer and used for the subsequent determination of DA and NE concentrations. The concentration of DA in the medium was very low or undetectable.

Statistical analysis of data.
Data are expressed as mean ± SEM and analyzed by 2-way ANOVA (using Sigma Plot Software, and GraphPad InStat, GraphPad Software, San Diego, CA; and SAS, SAS Institute Inc., Cary, NC). 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 test was set at {alpha} = 0.05. All comparisons were made with concomitantly run controls so that inter-assay variance would not confound the results.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of Chlorotriazine Metabolites on Cell Viability
The viability of cells in the HA-, deethylatrazine-, deisopropylatrazine-, and DACT-treated groups and their corresponding controls were expressed as the % change, and data are summarized from a representative experiment (Fig. 3Go). A 10–12% decrease in the viability was observed in PC12 cells treated with the higher concentrations of HA (200 and 400 µM) at 18 and 24 h. Deethylchlorotriazine, deisopropylchlorotriazine, and DACT only slightly reduced (2–5%) the viability following exposure to higher concentrations at longer time points. Thus, higher concentrations of HA may have had an impact on the catecholamines in these experiments; lower concentrations (6.25–100 µM HA) were effective in altering the catecholamine levels when viability was not altered. The effectiveness of deethylchlorotriazine, deisopropylchlorotriazine, and DACT in altering catecholamines was not related to an effect on cell viability.



View larger version (51K):
[in this window]
[in a new window]
 
FIG. 3. Viable cells from 6–24 h (% change) following treatment with hydroxyatrazine (HA), 2-amino-4-chloro-6-isopropylamino-s-triazine (deethylatrazine), 2-amino-4-chloro-6-ethylamino-s-triazine (deisopropylatrazine), and diaminochlorotriazine (DACT). PC12 cell cultures were exposed to HA with 0 µM, 100 µM, 200 µM, and 400 µM concentrations for the indicated time points. Similarly, PC12 cell cultures were treated respectively with deethylatrazine and deisopropylatrazine 0 µM, 50 µM, 100 µM, and 200 µM concentrations, respectively. They were also exposed to DACT with 0 µM, 40 µM, 80 µM, and 160 µM concentrations for the indicated time points. Each bar is the % change value of the mean of 6 cultures of a representative experiment. Data were duplicated in the subsequent experiment.

 
Effect of Hydroxyatrazine on the Intracellular DA and NE Concentrations and NE Release
The intracellular concentrations of DA and NE and the level of NE in the medium following exposure to hydroxyatrazine (HA) are shown in Figure 4Go. At each time point except at 3 h, there was a dose- and time-dependent decrease in the intracellular DA concentration. Similarly, intracellular NE concentration in these cells was reduced by exposure to 50 µM to 400 µM HA at 18 through 24 h. Norepinephrine release was also suppressed in a dose- and time-dependent manner. HA (200–400 µM) decreased the amount of NE released into the medium at 3 and 6 h, while 25–400 µM HA significantly reduced the release at 12 through 24 h.



View larger version (73K):
[in this window]
[in a new window]
 
FIG. 4. Time course of released NE and intracellular DA and NE concentrations following exposure to hydroxyatrazine (HA). PC12 cell cultures were exposed to HA with 0 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM, 200 µM, and 400 µM concentrations for the indicated time points. A represents the amount of NE released into the medium, while B and C represent the intracellular DA and NE levels, respectively. Each bar is the value of the mean ± SEM of >= 12 cultures. Data were duplicated in the subsequent experiment; * indicates the significance level (p < 0.05) at each time point when compared to the respective control.

 
Effect of Deethylchlorotriazine and Deisopropylchlorotriazine on the Intracellular DA and NE Concentrations and NE Release
Exposure to deethylchlorotriazine and deisopropylchlorotriazine resulted in a change in catecholamine synthesis that was different from that observed following HA. As shown in Figure 5Go, deethylchlorotriazine increased the concentration of intracellular DA following 100–200 µM at 12 h, and 50–200 µM at 18 through 24 h. A similar pattern of increased intracellular DA concentration was observed following exposure to deisopropylchlorotriazine at 200 µM at 6 h, 100–200 µM at both 12 and 18 h, and with 50–200 µM at 24 h (Fig. 6Go). Intracellular NE concentration was unchanged following exposure to both deethylchlorotrazine and deisopropylchlorotriazine at all concentrations through 18 h of exposure (Figs. 5 and 6GoGo). However, at 24 h, deethylchlorotriazine (50–200 µM) significantly reduced the intracellular NE concentration. NE release was also decreased by both deethylchlorotriazine and deisopropylchlorotriazine at 18 h (for deethylchlorotriazine) and 24 h (for both deethylchlorotriazine and deisopropylchlorotriazine) with doses of 100–200 µM (Figs. 5 and 6GoGo).



View larger version (81K):
[in this window]
[in a new window]
 
FIG. 5. Time course of released NE and intracellular DA and NE levels following exposure to 2-amino-4-chloro-6-isopropylamino-s-triazine (deethylatrazine). PC12 cell cultures were exposed to deethylatrazine with 0 µM, 12.5 µM, 25 µM, 50 µM, 100 µM, and 200 µM concentrations for the indicated time points. A represents the amount of NE released into the medium, while B and C represent the intracellular DA and NE levels, respectively. Each bar is the value of mean ± SEM of >= 12 cultures. Data were duplicated in subsequent experiment; * indicates the significance level (p < 0.05) at each time point, as compared to the respective control.

 


View larger version (74K):
[in this window]
[in a new window]
 
FIG. 6. Time course of released NE and intracellular DA and NE levels following exposure to 2-amino-4-chloro-6-ethylamino-s -triazine (deisopropylatrazine). PC12 cell cultures were exposed to deisopropylatrazine with 0 µM, 12.5 µM, 25 µM, 50 µM, 100 µM, and 200 µM concentrations for the indicated time points. A represents the amount of NE released into the medium, while B and C represent the intracellular DA and NE levels, respectively. Each bar is the value of the mean ± SEM of >= 2 cultures. Data were duplicated in the subsequent experiment; * indicates the significance level (p < 0.05) at each time point, as compared to the respective control.

 
Effect of Diaminochlorotriazine on the Intracellular DA and NE Concentrations and NE Release
Intracellular DA and NE concentrations and released NE concentrations following exposure to DACT are shown in Figure 7Go. The intracellular concentrations of DA and NE significantly increased following exposure to 160 µM DACT at 18 through 24 h, while NE release was significantly increased following exposure to 40–160 µM DACT after 24 h of treatment.



View larger version (76K):
[in this window]
[in a new window]
 
FIG. 7. Time course of released NE and intracellular DA and NE levels following exposure to diaminochlorotriazine (DACT). PC12 cell cultures were exposed to DACT with 0 µM, 10 µM, 20 µM, 40 µM, 80 µM, and 160 µM concentrations for the indicated time points. A represents the amount of NE released into the medium, while B and C represent the intracellular DA and NE levels, respectively. Each bar is the value of the mean ± SEM of >= 12 cultures, where experiments duplicated; * indicates the significance level (p < 0.05) at each time point, as compared to the respective control.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of these experiments are important for several reasons:

The different pattern of catecholamine metabolism observed after chlorotriazine exposure may provide some insight into which metabolites may be important enough to be involved in the reported in vivo responses. Specifically, hydroxyatrazine (HA) significantly decreased intracellular DA and NE levels, and in turn lowered NE release at the longer time points. This change in catecholamine concentrations is similar to that previously reported following exposure to the parent chlorotriazines, atrazine and simazine, using this same PC12 cell system (Das et al., 2000Go). The range of HA concentrations required to alter catecholamine synthesis and release was also consistent with the range of atrazine and simazine concentrations reported to effectively alter catecholamine metabolism. It should be noted that HA, at the higher concentrations (200 and 400 µM), altered the viability of the PC12 cells. However, the range of doses (6.25–100 µM) that was effective in significantly reducing intracellular DA and NE concentrations and NE release was well below these apparent toxic levels. The fact that HA produced changes in catecholamine synthesis and NE release similar to those observed following atrazine exposure might be expected, since PC12 cells express a high level of cytochrome b5 and NADH:cytochrome P450 reductase (Mapoles et al., 1993Go), which may metabolize atrazine primarily to HA in culture.

Another pattern of response was seen following DACT exposure, in which all these measures were either not different (<40 µM, 6 and 12 h) or increased (40–160 µM, 18 and 24 h). It is of interest that Sanderson et al. (2000), in evaluating the ability of chlorotriazines to induce aromatase activity in human adrenocortical carcinoma H295R cells, also found that the response of H295R cells to deisopropylchlorotriazine and deethylchlorotriazine was similar, while that to DACT was different (Sanderson et al., 2000Go). The two other chlorinated metabolites of atrazine and simazine, deethylchlorotriazine and deisopropylchlorotriazine, induced a third pattern of change. Both induced an increase in intracellular DA concentration and decreased intracellular NE and NE release. It is interesting that this pattern of response is similar to that observed in vivo following atrazine exposure (Cooper et al., 1998; see Table 1Go).


View this table:
[in this window]
[in a new window]
 
TABLE 1 Summary of Changes in Catecholamines following Exposure to Chlorotriazines and Their Metabolites
 
These results provide insight for analyzing or evaluating any in vivo effect of either chlorotriazines or their metabolites. Both in vivo and in vitro studies, using rat and human tissues, have shown that atrazine is metabolized primarily to the three chlorinated metabolites (Catenacci et al., 1990Go; Hanioka et al., 1999Go; Ikonen et al., 1988Go; Lang et al., 1997Go), while the hydroxylated metabolite is considered as a minor one (Hanioka et al., 1998Go). Hanioka, using an anti-rat CYP2C11, demonstrated that the major pathway for this metabolic process was cytochrome P450. Conjugation to glutathione was also shown to play a minor part in atrazine phase-II metabolism (Adams et al., 1990Go). Thus, the differences between the effect of the hydroxylated vs. chlorinated chlorotriazine metabolites may reflect the ability of the PC12 cells to produce primarily the hydroxylated form, whereas in vivo, the chlorinated metabolites are predominant.

Data from the present study demonstrate that the common core-structure of the chlorotriazines and the metabolites is essential for retaining the ability to alter the catecholamine concentrations in PC12 cells. Since the hydroxylated and chlorinated metabolites tested in the present study express different pharmacological effects, it is suggested that the attached side chains are the determining factor in how catecholamine synthesis is affected (i.e., either inhibition or stimulation) in PC12 cells. Although the precise impact of the chlorotriazine mediates on catecholamines in vivo remains to be determined, these observations would indicate that chlorotriazine-induced changes in vivo could be a key part of the neuroendocrine cascade responsible for altered reproductive development and altered reproductive processes in adulthood. A number of reports suggest that atrazine exposure in utero and/or in adult life causes altered developmental and adult reproductive processes (Cooper et al., 1996Go, 1999Go, 2000; Cummings et al., 2000Go; Eldridge et al., 1994Go, 1999Go; Infurna et al., 1988Go; Laws et al., 2000Go; Stevens et al., 1994Go; Stoker et al., 1999Go, 2000Go; Tennant et al., 1994Go; Wetzel et al., 1994Go).

In addition, in vivo studies provide evidence that atrazine is primarily metabolized to deethyl- and deisopropylatrazine within 2–3 h of administration (Catenacci et al., 1990Go; Ikonen et al., 1988Go; Timchalk et al., 1990Go). Our present findings also suggest that the effect of the chlorotriazines and their metabolites on reproductive function is additive. Both parent chlorotriazines and their metabolites are persistent in the environment (half-lives, 244 days) (Frank and Sirons, 1985Go; Parmeggiani, 1983Go; Santolucito and Nauman, 1992Go; Solomon et al., 1996Go), even though degradation by atzABC catabolic genes in atrazine-degrading bacteria can occur (De Souza et al., 1998Go; Mandelbaum et al., 1993Go).

In summary, like the chlorotriazines, the metabolites tested in this study are also capable of interacting with the sympathetic neuronal PC12 cells to alter the constitutive synthesis and release of catecholamines. Based on the LOEL of atrazine (12.5 µM) required to alter catecholamine synthesis in PC12 cells identified previously (Das et al., 2000Go), the present data indicate that the HA is more potent and deethyl- and deisopropylchlorotriazine are less potent than atrazine. Combined, our studies indicate that effective change in catecholamine synthesis and release occur with all chlorotriazines, with HA being the most potent and deethylchlorotriazine, deisopropylchlorotriazine, and diaminochlorotriazine the least (HA > atrazine > simazine > deethylchlorotriazine > deisopropylchlorotriazine > diaminochlorotriazine). The effect of HA exposure on catecholamine synthesis and release was very similar to that after atrazine and simazine exposure in these same cells (Das et al., 2000Go). In contrast, the observed effects following deethyl- and deisopropylchlorotriazine, but not the DACT exposure, is more like that observed in vivo after atrazine exposure (i.e., increased DA and decreased NE concentrations). Thus the pattern of metabolism may determine the particular effect on catecholamine synthesis and release. The primary focus of this study was to compare the pattern of responses between the parent chlorotriazines with that of their major metabolites in altering catecholamine synthesis and release in PC12 cells, which is a critical first step in characterizing the chlorotriazines and eventually determining their mechanism of action. Whether the cellular mechanism(s) of action for these effects on catecholamines is at the level of synthetic enzymes, intracellular processes, or the cell membrane remains to be determined.


    ACKNOWLEDGMENTS
 
This study has been funded wholly or in part by the U.S. Environmental Protection Agency. The authors acknowledge the assistance of Connie A. Meacham, Neurotoxicology Division, and Christy Lambright, Reproductive Toxicology Division, NHEERL, U.S. EPA, in PC12 cell culture. The authors also thank Dr. Susan C. Laws, Endocrinology Branch, Reproductive Toxicology Division, and Dr. William R. Mundy, Cellular and Molecular Toxicology Branch, Neurotoxicology Division, NHEERL, U.S. EPA, for their expert comments on the manuscript.


    NOTES
 
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. Fax: (919) 541-5138. E-mail: cooper.ralph{at}epa.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adams, N. H., Levi, P., and Hodgson, E. (1990). In vitro studies of the metabolism of atrazine, simazine, and terbutryn in several vertebrate species. J. Agric. Food Chem. 38, 1411–1417.[ISI]

Ahrens, W. H. (1994). Atrazine. In Herbicide Handbook, (W. H. Ahrens, Ed.), pp. 20–23. Weed Science Society of America, Champaign, Illinois.

Bakke, J., Larson, J. D., and Price, C. E. (1972). Metabolism of atrazine and 2-hydroxyatrazine by the rat. J. Agric. Food Chem. 20, 602–607.[ISI][Medline]

Barraclough, C. A. (1992). Neuronal control of the synthesis and release of luteinizing hormone-releasing hormone. Ciba Found. Symp. 168, 233–251.[ISI][Medline]

Bradway, D. E., and Moseman, R. F. (1982). Determination of urinary residue levels of the N-dealkyl metabolites of triazine herbicides. J. Agric. Food Chem. 30, 244–247.[ISI]

Ben-Jonathan, N. (1985). Dopamine: A prolactin-inhibiting hormone. Endocr. Rev. 6, 564–589.[ISI][Medline]

Budavari, S. (Ed.) (1996). The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals. Merck and Co., Inc., Whitehouse Station, NJ.

Catenacci, G., Maroni, M., Cottica, D., and Pozzoli, L. (1990). Assessment of human exposure to atrazine through the determination of free atrazine in urine. Bull. Environ. Contam. Toxicol. 44, 1–7.[ISI][Medline]

Cooper, R. L., McElroy, W. K., and Stoker, T. E. (2000b). Disruption of ovarian cycles by chlorotriazines and their metabolites in the rat. Toxocologist 54, 366.

Cooper, R. L., Goldman, J. M., Stoker, T. E. (1999). Neuroendocrine and reproductive effects of contemporary-use pesticides. Toxicol. Ind. Health 15, 26–36.[ISI][Medline]

Cooper, R. L., Stoker, T. E., McElroy W. K. and Hein, J. (1998). Atrazine (ATR) disrupts hypothalamic catecholamines and pituitary function. Toxicologist 42, 160.

Cooper, R. L., Stoker, T. E., Goldman, J. M., Parrish, M. B., and Tyrey, L. (1996). Effect of atrazine on ovarian function in the rat. Reprod. Toxicol. 10, 257–264.[ISI][Medline]

Cooper, R. L., Stoker, T. E., Tyrey, L., Goldman, J. M., and McElroy, W. K. (2000a). Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicol. Sci. 53, 297–307.[Abstract/Free Full Text]

Cummings, A. M., Rhodes, B. E., and Cooper, R. L. (2000). Effect of atrazine on implantation and early pregnancy in four strains of rats. Toxicol Sci. 58, 135–143.[Abstract/Free Full Text]

Das, P. C., McElroy, W. K., and Cooper, R. L. (2000). Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro. Toxicol. Sci. 56, 324–331.[Abstract/Free Full Text]

De Souza, M. L., Seffernick, J., Martinez, B., Sadowsky, M. J., and Wackett, L. P. (1998). The atrazine catabolism genes atzABC are widespread and highly conserved. J. Bacteriol. 180, 1951–1954.[Abstract/Free Full Text]

Eldridge, J. C., Tennant, M. K., Wetzel, L. T., Breckenridge, C. B., and Stevens, J. T. (1994). Factors affecting mammary tumor incidence in chlorotriazine-treated female rats: Hormonal properties, dosage, and animal strain. Environ. Health Perspect. 102, 29–36.

Eldridge, J. C., Wetzel, L. T., Stevens, J. T., and Simpkins, J. W. (1999). The mammary tumor response in triazine-treated female rats: A threshold-mediated interaction with strain- and species-specific reproductive senescence. Steroids 64, 672–678.[ISI][Medline]

Erickson, M. D., Frank, C. W., and Morgan, D. P. (1979). Determination of s-triazine herbicide residues in urine: Studies of excretion and metabolism in swine as a model to human metabolism. J. Agric. Food Chem. 27, 743–746.[ISI][Medline]

Flury, M. (1996). Experimental evidence of transport of pesticides through field soils (review). J. Environ. Qual. 25, 25–45.[ISI]

Frank, R., and Sirons, G. J. (1985). Dissipation of atrazine residues from soils. Bull. Environ. Contam. Toxicol. 34, 541–548.[ISI][Medline]

Goldman, J. M., Stoker, T. E., Cooper, R. L., McElroy, W. K., and Hein, J. F. (1994). Blockade of ovulation in the rat by the fungicide sodium N-methyldithiocarbamate: Relationship between effects on the luteinizing hormone surge and alterations in hypothalamic catecholamines. Neurotoxicol. Teratol. 16, 257–268.[ISI][Medline]

Greene, L. A., and Tischler, A. S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U.S.A.. 73, 2424–2428.[Abstract]

Greene, L. A., and Tischler, A. S. (1982). PC12 pheochromocytoma cultures in neurobiological research. Adv. Cell. Neurobiol. 3, 373–414.

Gudderwar, M. B., and Dauterman, W. C. (1979). Studies on a glutathione S-transferase preparation from mouse liver which conjugates chloro-s-triazine herbicides. Pestic. Biochem. Physiol. 12, 1–9.[ISI]

Hanioka, N., Jinno, H., Kitazawa, K., Tanaka-Kagawa, T., Nishimura, T., Ando, M., and Ogawa, K. (1998). In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes. Chem. Biol. Interact. 116, 181–198.[ISI][Medline]

Hanioka, N., Jinno, H., Tanaka-Kagawa, T., Nishimura, T., Ando, M. (1999). In vitro metabolism of simazine, atrazine, and propazine by hepatic cytochrome P450 enzymes of rat, mouse ,and guinea pig, and oestrogenic activity of chlorotriazines and their main metabolites. Xenobiotica 29, 1213–1226.[ISI][Medline]

Harris, C. I. (1967). Fate of 2-chloro-s-triazine herbicides in soil. J. Agric. Food Chem. 15, 157–162.[ISI]

Ikonen, R., Kangas, J., and Savolainen, H. (1988). Urinary atrazine metabolites as indicators for rat and human exposure to atrazine. Toxicol. Lett. 44, 109–112.[ISI][Medline]

Infurna, R., Levy, B., Meng, C., Yau, E., Traina, V., Rolofson, G., Stevens, J., and Barnett, J. (1988). Teratological evaluations of atrazine technical, a triazine herbicide, in rats and rabbits. J. Toxicol. Environ. Health, 24, 307–319.[ISI][Medline]

Kalra, S. P., and Kalra, P. S. (1983). Neural regulation of luteinizing hormone secretion in the rat. Endocrine Rev. 4, 311–351.[ISI][Medline]

Kello, D. (1989). WHO drinking water quality guidelines for selected herbicides. Food Addit. Contam. 6, S79–S85.[ISI][Medline]

Koskinen, W. C., and Clay, S. A. (1997). Factors affecting atrazine fate in north central U.S. soils. Rev. Environ. Contam. Toxicol. 151, 117–165.[Medline]

Lamoureux, G. L., Simoneaux, B., and Larson, J. (1998). The metabolism of atrazine and related 2-chloro-4,6-bis(alkylamino)-s-triazines in plants. In Triazine Herbicides: Risk Assessment, (L. G. Ballantine, J. E. McFarland and D. S. Hacket, Eds.), pp. 60–81. American Chemical Society, Washington, D.C.

Lang, D. H., Rettie, A. E., and Bocker, R. H. (1997). Identification of enzymes involved in the metabolism of atrazine, terbuthylazine, ametryne, and terbutryne in human liver microsomes. Chem. Res. Toxicol. 10, 1037–1044.[ISI][Medline]

Laws, S. C., Ferrell, J. M., Stoker, T. E., Schmid, J., and Cooper, R. L. (2000). The effects of atrazine on puberty in female Wistar rats: An evaluation of the protocol for the assessment of pubertal development and thyroid function. Toxicol. Sci. 58, 366–376.[Abstract/Free Full Text]

Levy, J., and Chesters, G. (1995). Simulation of atrazine and metabolite transport and fate in a sandy-till aquifer. J. Contam. Hydro. 20, 67–88.[ISI]

Ma, L., and Selim, H. M. (1996). Atrazine retention and transport in soils. Rev. Environ. Contam. Toxicol. 145, 129–173.[ISI][Medline]

Mandelbaum, R. T., Wackett L. P., and Allan, D. L. (1993). Rapid hydrolysis of atrazine to hydroxyatrazine by soil bacteria. Environ. Sci. Technol. 27, 1943–1946.[ISI]

Mapoles, J., Berthou, F., Alexander, A., Simon, F. and Menez, J.-F. (1993). Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression. Eur. J. Biochem. 214, 735–745.[Abstract]

Muir, D., and Baker, E. B. (1978). The disappearance and movement of three triazine herbicides and several of their degradation products in soil under conditions. Weed Res. 18, 111–120.[ISI]

Parmeggiani, L. (1983). Encyclopedia of Occupational Health and Safety, 3rd ed. International Labour Office, Geneva.

Pinter, A., Torok, G., Borzsonyi, M., Surjan, A., Csik, M., Kelecsenyi, Z., and Kocsis, Z. (1990). Long-term carcinogenecity bioassay of the herbicide atrazine in F344 rats. Neoplasma 37, 533–544.[ISI][Medline]

Ramirez, V. D., Feder, H. H., and Sawyer, C. H. (1984). The role of brain catecholamines in the regulation of LH secretion: A critical inquiry. In Frontiers in Neuroendocrinology. (L. Martini and W. F. Ganong, Eds.), Vol. 8, pp. 27–84. Raven Press, New York.

Rodriguez, C. J., and Harkin, J. M. (1997), Degradation of atrazine in subsoils, and groundwater mixed with aquifer sediments. Bull. Environ. Contam. Toxicol. 59, 728–735.[ISI][Medline]

Sanderson, J. T., Letcher, R. J., Heneweer, M., and van den Berg, M. (2000). Effects of chloro-s-triazine herbicides and metabolites on aromatase (CYP19) activity in various human cell lines and on vitellogenin production in male carp hepatocytes. Toxicologist 54, 240.

Santolucito, J. A., and Nauman, C. H. (1992). Compendium of body burden biomarkers for pesticides. In EPA600/X-92/052, pp. 17–19. U.S. Environmental Protection Agency, Las Vegas.

Seiler, A., Brenneisen, P., and Green, D. H. (1992). Benefits and risks of plant protection products—possibilities of protecting drinking water: Case atrazine. Water Supply 10, 31–42.

Solomon, K. R., Baker, D. B., Richards, R. P., Dixon, K. R., Klaine, S. J., La Point, T. W., Kendall, R. J., Weisskopf, C. P., Giddings, J. M., Giesy, J. P., Hall, L. W., Jr., and Williams, W. M. (1996). Ecological risk assessment of atrazine in North American surface waters. Environ. Toxicol. Chem. 15, 31–76.[ISI]

Sorenson, B. A., Wyse, D. L., Koskinen, W. C., Buhler, D. D., and Jorgenson, M. D. (1993). Formation and movement of 14C-atrazine degradation products in a sandy loam soil under field conditions. Weed Sci. 41, 239–245.[ISI]

Stevens, J. T., Breckenridge, C. B., Wetzel, L. T., Gillis, J. H., Luempert, L. G. III, and Eldridge, J. C. (1994). Hypothesis for mammary tumorigenesis in Sprague-Dawley rats exposed to certain triazine herbicides. J. Toxicol. Environ. Health 43, 139–154.[ISI][Medline]

Stoker, T. E., Guidici, D. L., McElroy, W. K., Laws, S. C., and Cooper, R. L. (2000). The effect of atrazine (atr) on puberty in male Wistar rats. Toxicologist 54, 366.

Stoker, T. E., Robinette, C. L., and Cooper, R. L. (1999). Maternal exposure to atrazine during lactation suppresses sucking-induced prolactin release and results in prostatitis in the adult offspring. Toxicol. Sci. 52, 68–79.[Abstract]

Tennant, M. K., Hill, D. S., Eldridge, J. C., Wetzel, L. T., Breckenridge, C. B., and Stevens, J. T. (1994). Chloro-s-triazine antagonism of estrogen action: Limited interaction with estrogen receptor binding. J. Toxicol. Environ. Health 43, 197–211.[ISI][Medline]

Thakur, A. K., Wetzel, L. T., Tisdel, M. O., and Stevens, J. T. (1992). Comparison of the potential effects of atrazine on the development of mammary, pituitary, uterine, and ovarian tumors in Sprague-Dawley and Fisher 344 female rats. Toxicologist 12, 380.

Timchalk, C., Dryzga, M. D., Langvardt, P. W., Kastl, P. E., and Osborne, D. W. (1990). Determination of the effect of tridiphane on the pharmakokinetics of [14C]-atrazine following oral administration to male Fischer-344 rats. Toxicology 61, 27–40.[ISI][Medline]

U.S. EPA (U.S. Environmental Protection Agency) (1994). Atrazine, Simazine, and Cyanazine; Notice of Initiation of Special Review. 59 Fed. Regist., 60412–60443.

Wetzel, L. T., Luempert, L. G., III, Breckenridge, C. B., Tisdel, M. O., Stevens, J. T., Thakur, A. K., Extrom, P. J., and Eldridge, J. C. (1994). Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fisher 344 rats. J. Toxicol. Environ. Health 43, 169–182.[ISI][Medline]

Winkelmann, D. A., and Klaine, S. J. (1991). Degradation and bound residue formation of four atrazine metabolites, deethylatrazine, deisopropylatrazine, dealkylatrazine and hydroxyatrazine, in a western Tennessee soil. Environ. Toxicol. Chem. 10, 347–354.[ISI]

Worthing, C. R., and Walker, S. B. (1987). The Pesticide Manual: A World Compendium. British Crop Protection Council, Thornton Heath, England.