* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; and
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
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ABSTRACT |
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Key Words: chlorotriazines; atrazine; simazine; cyanazine; PC12 cell line; dopamine; norepinephrine..
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INTRODUCTION |
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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, 1992; Kalra and Kalra, 1983
; Ramirez et al., 1984
). Likewise, increasing hypothalamic DA turnover will lower PRL secretion by the pituitary (Ben-Jonathan, 1985
). 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, 1986
; Akema et al., 1990
; Fuchs et al., 1984
; Kang et al., 1995
; Leonhardt et al., 1995
). 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, 1976). 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, 1976
). 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.
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MATERIALS AND METHODS |
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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, 1976). 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,
-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, 1976
) 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 9798% 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 48% 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., 1994) using a mobile-phase buffer containing 48% 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 1. 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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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 = 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.
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RESULTS |
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DISCUSSION |
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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., 1997; Kim et al., 1993
). This effect on gene expression is mediated through the phosphorylation of cAMP response element binding protein (CREB) (Dash et al., 1991
; Sheng et al., 1991
). 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, 1993
; Kim et al., 1993
, 1994
; Lewis et al., 1987
). 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., 1993
; Tank et al., 1986
). 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, 1986; Akema et al., 1990
; Fuchs et al., 1984
; Kang et al., 1995
; Leonhardt et al., 1995
). PC12 cells possess GABAA-R Cl ion channels, and the GABAA-R subunits identified in PC12 cells include ß3,
2L,
2s, and
subunits (Tyndale et al., 1994
). These cells also contain the benzodiazepine-binding site (Miller et al., 1988
; Morgan et al., 1985
). Interestingly, these cells do not respond to GABA-ergic stimulation (Hales and Tyndale, 1994
; Wisden, Moss, 1997
) 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 2.6943.14 ppm for atrazine, 2.5240.34 ppm for simazine, and 6.0148.14 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 70400 ppm (Stevens et al., 1994). Pubertal exposure to atrazine was found to delay preputial separation (LOAEL = 12.5 mg/kg or ~175 ppm) (Stoker et al., 2000
). 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., 1999
). 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., 1994
). The order of potency for these in vivo effects in the female rat following oral exposure is cyanazine > atrazine
simazine (Eldridge et al., 1994
; Cooper et al., 2000
).
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, 1992; Cooper et al., 1999
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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