* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; and
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
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ABSTRACT |
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Key Words: chlorotriazine metabolites; hydroxyatrazine; deethylchlorotriazine; deisopropylchlorotriazine; diaminochlorotriazine; PC12 cells; dopamine; norepinephrine.
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INTRODUCTION |
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The metabolites identified in the environment and in the urine of mammals (including humans) are presented in Figure 1. In the environment, one of the major atrazine degradation pathways is chemical hydrolysis to 6-hydroxyatrazine in the surface soil (Harris, 1967
; Muir and Baker, 1978
), something that is favored by low pH (5.56.5), elevated moisture content, high temperature, and increased organic matter levels (Ahrens, 1994
; Koskinen and Clay, 1997
; Ma and Selim, 1996
). 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., 1998
; Mandelbaum et al., 1993
). 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, 1994
; Koskinen and Clay, 1997
; Ma and Selim, 1996
). The microbial degradation of chlorinated products of atrazine has higher mobility and greater potential to contaminate groundwater than hydroxyatrazine (Sorenson et al., 1993
), which is strongly adsorbed onto surface soils (Ma and Selim, 1996
). 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, 1991
), whereas in a sandy-till aquifer, the half-lives of these metabolites is considerably longer, ranging from 2700 to 3400 days (Levy and Chesters, 1995
). 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, 1997
), and persists for a long time in the environment, potentially affecting human and wildlife populations.
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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 2. Recent studies in our laboratory indicate that several of these metabolites, administered in vivo, affect ovarian cyclicity (Cooper et al., 2000b
), 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, 1976
, 1982
) to examine whether the major chlorotriazine metabolites alter the constitutive synthesis of dopamine and norepinephrine.
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MATERIALS AND METHODS |
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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, 1976). 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., 2000
). 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., 2000). 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 1.2378.88 ppm for HA, 2.3437.4 ppm for deethylchlorotriazine, 2.1634.6 ppm for deisopropylchlorotriazine, and 1.4555.29 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., 2000
). 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 9798% 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., 1994) 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 = 0.05. All comparisons were made with concomitantly run controls so that inter-assay variance would not confound the results.
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RESULTS |
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DISCUSSION |
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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., 2000). 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 (6.25100 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., 1993
), 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 (40160 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., 2000). 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 1
).
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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., 1996, 1999
, 2000; Cummings et al., 2000
; Eldridge et al., 1994
, 1999
; Infurna et al., 1988
; Laws et al., 2000
; Stevens et al., 1994
; Stoker et al., 1999
, 2000
; Tennant et al., 1994
; Wetzel et al., 1994
).
In addition, in vivo studies provide evidence that atrazine is primarily metabolized to deethyl- and deisopropylatrazine within 23 h of administration (Catenacci et al., 1990; Ikonen et al., 1988
; Timchalk et al., 1990
). 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, 1985
; Parmeggiani, 1983
; Santolucito and Nauman, 1992
; Solomon et al., 1996
), even though degradation by atzABC catabolic genes in atrazine-degrading bacteria can occur (De Souza et al., 1998
; Mandelbaum et al., 1993
).
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., 2000), 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., 2000
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 541-5138. E-mail: cooper.ralph{at}epa.gov.
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