Testing Status of Agents at NTP
Executive Summary Atrazine
G. Other Toxicological Effects
- Endocrine Toxicology
In addition to the studies described below, endocrine studies are currently being conducted by Ciba-Geigy to investigate a possible endocrine basis for atrazine-related mammary tumor formation in female Sprague-Dawley rats (see section V.D.2). According to Ciba-Geigy, the preliminary data suggest that exposure of female Sprague-Dawley rats to high levels of atrazine may accelerate an existing endocrine imbalance that is naturally conducive to the development of mammary tumors. Results from these ongoing studies (on female Sprague-Dawley rats) include the following [Ciba-Geigy, 1991]:
- - Circulating serum hormone levels,
including estrogen and progesterone, are altered.
- - Estrous cycle duration and pattern are changed.
- - A reduction in the uterotropic response to estrogen has been noted, indicating a possible anti-estrogenic effect in that tissue.
- - Estrogen receptor binding affinity is altered in the hypothalamus, pituitary, mammary gland, and uterus. Similar effects are noted in vitro with hypothalamic, pituitary, and uterine receptors.
- - Interference with estrogen receptor binding appears to be competitive (i.e., the affinity of estrogen for binding to its receptor is decreased while specific binding capacity is unaffected).
- - Estrous cycle duration and pattern are changed.
The effect of dietary atrazine on the in-vivo synthesis of prostaglandin E1 (PGE1) and thromboxane B2 (TXB2) by platelets from clotted blood was studied in 1-month-old male Wistar rats. Groups of rats were administered diets containing 500 mg atrazine/kg diet for 30-35 days, and serum PGE1 and TXB2 activity was determined by radioimmunoassay. The authors concluded that the effect of atrazine on the synthesis of PGE1 and TXB2 was not statistically significant (P<0.05) [Meydani et al., 1984].
oral, rats/rat anterior pituitary and hypothalamus cells
The effects of atrazine and deethylatrazine (a primary metabolite of atrazine) on the enzymic systems responsible for testosterone metabolism in the anterior pituitary and hypothalamus were studied. Male Fisher rats were orally dosed with 6 or 12 mg/100g atrazine or deethylatrazine for 7 days. At the 12 mg/100g dose level, atrazine significantly (P< 0.01) increased the weight of the anterior pituitary with hyperemia and hypertrophy of chromophobic cells with vacuolar degeneration. At this dose, 5 alpha reductase (alpha-R) activity (P<0.01/0.02), and 3 alpha- and 17 beta-HSD activities (P<0.01) were inhibited in the anterior pituitary. The 12 mg/100 g dose of deethylatrazine significantly inhibited 5 alpha-R activity in the anterior pituitary (P<0.01). The authors suggested that, based on the similar reduction in 5 alpha-R activity caused by both atrazine and deethyl atrazine, the mechanism of inhibition may be similar. No changes in 3 alpha- and 17 beta-hydroxysteroid dehydrogenase (HSD) activities were observed in the high dose group compared to the controls. The lower dose of atrazine and deethylatrazine did not influence the enzymic activities involved in testosterone conversion in the anterior pituitary. At the 12 mg/100 kg dose, atrazine significantly inhibited 5 alpha-R (P< 0.02) and 17 beta-HSD (P<0.01) activity in the hypothalamus tissue. At the 6 mg/100 kg dose, atrazine significantly (P<0.01) decreased 17 beta-HSD activity in the male rat hypothalamus. Deethylatrazine, at a dose of 12 mg/100g, significantly inhibited 5 alpha-R (P<0.01) and 17 beta-HSD (P<0.01) activities in the hypothalamus.
In addition to the oral study, an in vitro study on male Fisher rat anterior pituitary and hypothalamus cells was performed concurrently. Atrazine or deethylatrazine, was added into the incubation medium at a dose of .92 µM atrazine or deethylatrazine in 5 µl ethanol added into 2 ml of incubation medium. Addition of atrazine or deethylatrazine, significantly inhibited (P < 0.01) 5 alpha-R, 3 alpha- and 17 beta-HSD activities in the anterior pituitary. The inhibition of 5 alpha-R activity was more marked for atrazine. The in vitro addition of deethylatrazine was more effective in inhibiting 5 alpha-R, 3 alpha- and 17 beta-HSD (P < 0.01) in the male rat hypothalamus than atrazine. Atrazine was shown only to inhibit 3 alpha-HSD activity (P < 0.01) in the hypothalamus [Babic-Gojmerac et al., 1989]. In an earlier study, Kniewald and Kniewald demonstrated that a mixture of atrazine and prometryne (0.56 mmol of each of the compounds) inhibited the formation of the active androgen metabolite in the male rat pituitary by 77% [Kniewald and Kniewald, 1983].
subcutaneous, rats
Kniewald et al., studied the effect of atrazine and its metabolite, deethylatrazine, on the rat gonadotropic mechanism during the early postnatal period. An unspecified number of pregnant female fisher rats (~180 g) were treated subcutaneously with atrazine or deethylatrazine (in 0.1 ml paraffin oil/animal) at a concentration of 1.66 mg/100 g/ body weight/day from day 1 of pregnancy throughout gestation. A second group of pregnant rats was treated with this dosing regimen throughout pregnancy and during lactation. Nontreated and oil injected controls were run for each group. Body weights of the treated and control mothers as well as their offspring were recorded weekly. Offspring up to 28 days of age from both groups of does fed from their own mothers. Male and female offspring were sacrificed on either day 21 or 28 of life, their anterior pituitaries were removed, and the ventral prostrate or uterus tissues were collected from each group for cytosol preparation. The number of specific binding sites for the dihydrotestosterone (DHT)-receptor in the prostrate and estradiol-receptor in the uterus were determined. To assess pituitary enzymatic activities, the conversion of [4-14C]-testosterone to the metabolites 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol); 5 alpha-dihydro testosterone (5 alpa-DHT); androstat-4-ene-3,17-dione; and 5 alpha-androstane 3,17-dione was studied. No significant differences in maternal body weight or the body weight of offspring from either group were observed.
Daily atrazine and deethylatrazine injections to rat mothers during pregnancy did not induce any significant changes in pituitary 5 alpha-reductase, 3-oxy-steroid 4-ene-dehydrogenase C (5 alpha-R); 3 a-hydroxysteroid dehydrogenase (3 alpha-HSD), or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) among 28-day old male offspring. Significantly (P<0.05 {atrazine}) increased levels of 5 alpha-R activity were observed among female offspring. Kniewald et al., report that this data provides evidence that treatment of mothers during pregnancy induces, through the placenta, long-term effects in female offspring.
Treatment of does during both pregnancy and lactation with the two compounds induced a significant (P<0.01{atrazine}, P<0.05{deethylatrazine)) decrease in the formation of (3 alpha-diol) in 21-day-old male offspring. In addition, (5 alpha-DHT) formation was significantly (P<0.05) decreased by atrazine in 21-day-old males. Androstat-4-ene-3,17-dione and 5 alpha-androstane 3,17-dione formation were not affected by treatment with either chemical in male offspring. However, 21-day-old female offspring did exhibit significantly (P<0.05) decreased levels of androstat-4-ene-3,17-dione. The authors noted that while there were no differences in the weights of the pituitaries, these observed decreases in enzymatic activity were a direct result of treatment of mothers during pregnancy and lactation.
Concerning the results of hormone-receptor specific binding site determination in gonads of the 28-day-old offspring, treatment of dams with atrazine and deethylatrazine during pregnancy did not change the number of specific binding sites for the prostate DHT-receptor or for the uterus estradiol receptor. However, treatment with these compounds during both pregnancy and lactation induced a significant (P value not reported) reduction in the number of specific binding sites for prostate DHT-receptors in 21-day-old offspring. The authors conclude that, in rats, exposure to atrazine and its metabolite during pregnancy only, or during pregnancy and lactation, influences the pituitary-gonadal axis of male and female offspring [Kniewald et al., 1987].
unspecified route, rats
Male Wistar rats were treated with 50 ppm/day atrazine (route not specified) for 15 days. Body, thyroid, adrenal and hypophysis weight were studied, and blood samples were collected for T3, T4 and lactic hydrogenase (LH) assays. Atrazine caused a slight decrease in thyroid weight, body weight, and weight of the hypophysis. T3 and T4 release in the serum was stimulated by atrazine while LH release in the serum was inhibited by atrazine. The amount of LH in the hypophysis showed an increase under the action of atrazine (P values not specified in study) [Ghinea et al., 1979].
thyroid cells , humans
The effect of atrazine was studied in human thyroid cell cultures human thyroid to determine its influence on cell multiplication, protein synthesis, enzymatic activity of thyroid cells, and hormonal synthesis. Cultured cells were obtained by trypsinization from normal and pathological (Graves disease, thyroid nodule) human tissue which had been removed by surgery. Cell cultures were treated with doses of 0.0001, 0.001, 0.01, and 0.1 mg atrazine per culture tube containing 3 x 105 cells/1.5 ml medium. In normal cells, atrazine caused a strong inhibition of protein synthesis. In cell cultures of thyroid nodule, atrazine showed a slight stimulating effect on protein synthesis, and in Graves disease cultures, no overall effect was noted. Effect on cell multiplication was similar to that for protein synthesis. At the 0.1 mg dose in the normal cells, atrazine caused cellular changes, cytoplasmic vacuoles, widely spaced nuclei, and thin-layered and extended cytoplasm. The Graves disease cells showed a decreased lactic dehydrogenase activity for all doses of atrazine. Atrazine caused inhibition of peroxidases at the high doses. Atrazine also caused an inhibition of RNA synthesis in all of the thyroid cell types. In the tube containing normal cells treated with 0.01 or 0.1 mg atrazine per tube, T3 and T4 cells were radioimmunologically assayed. Radioimmunologic assay of T3 and T4 cells in the culture medium of normal thyroid cells showed a slight stimulation of secretion of T3 and T4 cells induced by atrazine (P values not specified in study)[Ghinea et al., 1979].
embryo renal cells, humans
The influence of atrazine on the conversion of T4 to T3 cells in human embryo renal cell cultures was studied. Human embryo renal cells were dosed with 0.1 or 1.0 µg/ml of atrazine, with varying levels of T4, and with and without cysteine. Atrazine significantly decreased T3 release (P = 1%) at both doses of T4 [Ghinea et al., 1986].
thyroid cancer cells, humans
The reactivity of thyroid cancer cells treated with estradiol and dehydroepiandrosterone (DHA) in the presence of other hormones (TSH, STH, insulin), myopeptides and pesticides, including atrazine, was studied. It was found that DHA exerted an inhibitory effect on protein synthesis, and that this effect was increased by the presence of atrazine. Also, release of Tg1 in the culture medium was inhibited by atrazine ( P values not specified) [Ghinea et al., 1988].
renal and liver cells, rats
The influence of atrazine on the conversion of T4 to T3 cells in kidney and liver cell cultures, with and without cysteine present, was studied. Rat renal cell cultures and organotypic hepatic tissue cultures were dosed with 0.1 or 1.0 µg/ml of atrazine, with varying levels of T4 , and with and without cysteine. Treated cultures showed an increase in cell density for the high dose, proportional with the dose of T4 used. The enzymatic activity, the presence of lactic dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase was increased in lots receiving 10 µg T4 and atrazine (P value not specified, but increase was proportional to dose). In the rat renal cells, protein synthesis was inhibited by both doses of T4, but atrazine produced a significant stimulation (P value not specified, but increase is proportional to dose). In the rat renal cell cultures, atrazine significantly increased T3 release in the high T4 groups (P=5% and 1%, respectively in the presence and absence of cysteine). This is in contrast to the results found in the human embryo renal cells (see above) [Ghinea et al., 1986].
pituitary, calves
Male and female calf pituitaries were collected and assayed for pituitary enzymatic activity. It was shown that atrazine induces inhibition of the enzymic activity in the male and female calf pituitary (P values not specified) [Kniewald and Kniewald, 1982].
- - Circulating serum hormone levels,
including estrogen and progesterone, are altered.
- Immunotoxicity
oral, rats
The immunotoxicity of atrazine was studied in 4 groups of 6 weanling male Riv: TOX (M) Wistar rats. A semisynthetic diet containing 0, 100, 300, or 900 mg/kg atrazine (97% technical) was provided ad libitum for three weeks. Body weight and food intake were recorded weekly. At the end of the 3-week period, exsanguination was done from the aorta, and the weights of the liver, kidney, pituitary, adrenals, thyroid, testes, thymus, spleen, and mesenteric and popliteal lymph nodes were determined. Samples of these organs were also fixed and stained. Finally, total leukocyte and differential leukocyte counts were carried out and serum IgM and IgG were quantified.
Atrazine was found to cause a significant decrease in terminal body weight (P<0.01) and daily food intake (P<0.001) in the 900 mg/kg dose group only. The serum lymphocyte concentration of rats in the 100 mg/kg (P<0.05), 300 mg/kg (P<0.05), and 900 mg/kg (P<0.01) dose groups was also significantly reduced. Weights of the thyroid (P<0.01) and mesenteric lymph node (P<0.05) were significantly increased in the 900 mg/kg dose group only, while the weight of the thymus was significantly (P<0.05) decreased in this dose group. Histopathological effects, which were observed in only 1 animal from the high dose group, included homogeneous cytoplasm hepatocytes, slight cortex atrophy of the thymus, and slight depletion of PALS in the spleen. The authors concluded the alteration of immunological parameters was a sensitive indicator of atrazine-induced toxicity [Vos et al., 1983].
- Neurotoxicity
unspecified route, human
In 1980, a farmer suffering from a sensorimotor polyneuropathy disabling his lower extremities was admitted to a clinic in Milan, Italy. These symptoms of neurotoxicity were attributed to atrazine exposure. Although no other data was reported, it was stated that this incident prompted Castano et al., to study the potential neurotoxic effects of atrazine in rats (see below) [Castano et al., 1982].
oral, rat
The neurotoxicological effects of technical grade atrazine (95% purity) were studied in 10 CFY male rats by dietary administration at concentrations 1/20 and 1/40 of the LD50 (75 mg/kg/day and 37.5 mg/kg/day, respectively) for 6 weeks. Behavioral experiments were carried out in a maze consisting of four T-shaped elements. After 15-hours of food deprivation, the rats were put into the starting point and their running time to the goal and the number of errors made were measured daily during the 6-week testing period. Atrazine was not found to affect either behavioral parameter [Dési, 1983].
intraperitoneal, rat
Castano et al., investigated the neurotoxic effects of atrazine in 40 Sprague-Dawley rats which were divided into 3 groups. Animals in groups 1 and 2 (n=14/group) received 40 mg and 20 mg of atrazine, respectively dissolved in 1 ml of dimethyl sulfoxide by intraperitoneal injection. A third group (n=12), which served as the control, was administered dimethyl sulfoxide only. For each group, atrazine, or the vehicle control, was injected twice weekly for a total period of 30 days. Half of the animals in each group were sacrificed at the end of treatment and the other half were sacrificed after a 30-day recovery period. Sciatic nerve, spinal ganglia, and spinal cord samples were removed and examined by electron microscopy. In addition, samples of the liver, kidney, lung and lymph nodes were examined microscopically.
The only effect observed was a significant decrease in axonal areas, both in myelinated (P>0.05) and unmyelinated (P>0.01) fibers among rats treated with 40 mg atrazine and sacrificed immediately following treatment. After a recovery period of 30 days, axonal areas had returned to normal. No pathological changes were noted in the liver, kidney, lung, or lymph node. The authors concluded that high doses of atrazine have a reversible neurotoxic effect, primarily involving unmyelinated fibers [Castano et al., 1982].
- Biochemical Toxicology
oral, rats
The acute enzymotoxic effects of atrazine were examined in groups of 8 female Wistar rats following two oral doses of 0.75 g/kg atrazine in a glycerol-water mixture administered two weeks apart (on days 1 and 14). Control animals received equal doses of the vehicle. Animals were sacrificed 2 and 6 hours, and 1, 2, 3, 5, 7, and 14 days after the first dose, and 1, 3, and 7 days after the second dose. Liver, spleen, and kidney homogenates were prepared for the determination of ceruloplasmin and acid phosphatase activity. In the liver, a sharp decrease in ceruloplasmin activity and a simultaneous mild increase of acid phosphatase activity occurred after the first dose, whereas the second dose caused a sudden rise of both enzymatic levels. In the kidney, ceruloplasmin decreased and acid phosphatase slightly increased on the 2nd and 3rd days after the first dose; however, the second dose caused an increase in ceruloplasmin and a very slight decrease in acid phosphatase levels. In the spleen, the levels of ceruloplasmin generally remained within the limits of the control values following both doses. Acid phosphatase activity, however, significantly increased (P value not reported) after the first dose, and then was rapidly restored to normal levels by the second dose. The authors concluded that atrazine shows different effects on the activities of the enzymes studied, and that the liver was the most sensitive organ to the influence of this compound. The authors also state that the results seen following the second dose indicate that atrazine undergoes rapid degradation and elimination [Worth, et al., 1982].
oral, rats
Male and female Wistar rats were used to examine the effects of atrazine on organ glucose-6-phosphate dehydrogenase (G-6-PDH) and aldolase (ALD) activities following two oral doses of 750 mg atrazine/kg two weeks apart (on days 1 and 14). Atrazine was administered in 2 ml of a glycerol-water mixture. Control animals received equal doses of the vehicle. Animals were sacrificed 2 and 6 hours, and 1, 2, 3, 5, 7, and 14 days after the first dose, and 1, 3, and 7 days after the second dose. Liver, spleen, and kidney homogenates were prepared for the determination of enzyme activity. The results demonstrated that G-6-PDH activity decreased in the liver only 2 hours after the first dose of atrazine, but returned to the normal by day 14. In both the kidney and the spleen, the first dose had no effect on the G-6-PDH activity, and in all three organs, the G-6-PDH activity was lowered on the first day after the second dose and then returned to normal after 3 days. There was no apparent change in the ALD activity during either dose in the liver and the spleen; however, in the kidney, the second dose produced a marked decrease in ALD activity within one day of administration. The authors concluded that the quick recovery of enzyme activities to normal levels was presumably due to rapid elimination of atrazine. Also, a second dose administered after recovery from the first, produced transient enzymotoxic effects [Radovcic et al., 1978].
oral, rats
In a study examining the effects of atrazine on 5 alpha-dihydrotestosterone (DHT) receptor complex formation, groups of 8-12 male Fischer rats (28- and 90-days-old) were given daily oral doses of atrazine (12 mg/100 g body weight) in paraffin oil for 7 consecutive days. Control animals were given the same volume of vehicle. Animals were sacrificed 1-22 days after treatment, the prostrate gland was removed and homogenized, and the samples were analyzed for cytosol DHT receptors. Atrazine was found to inhibit DHT receptor complex formation in both 28- and 90-day-old animals; however, the inhibition was stronger in the young rats than in the sexually mature adults. The effect of atrazine on this process was reversible, and returned to normal 22 and 14 days after dosing was stopped in young and adult rats, respectively [Simic et al., 1991]
oral, rats
The effect of atrazine on monoamine oxidase (MAO) activity was studied in male Wistar rats. Groups of 7-12 animals were given daily oral doses of 220 mg atrazine/kg for 6 days, and MAO activity was determined in brain and liver tissues 2 hours after the sixth dose. Treatment with atrazine significantly inhibited (P<0.01) MAO activity in the brain, and significantly increased (P<0.01) the activity in the liver [Bainova et al., 1979].
oral/intraperitoneal, rats
The effects of atrazine on rat liver glycogen metabolism were investigated in female Sprague-Dawley rats following oral or intraperitoneal administration. Animals were either fed a diet containing 0.05% atrazine (0.3g/20 ml acetone mixed with 600g of standard diet) for 4 to 7 days, or were given an intraperitoneal injection of 25 mg atrazine (suspended in corn oil)/100 g rat. Control animals were fed a diet that did not contain atrazine or were administered injections of corn oil. Cyclic AMP activity was determined in the liver at unspecified times after oral dosing and at 5, 10, and 15 hours after the intraperitoneal dose. Liver glycogen phosphorylase, adenylate cyclase and phosphodiesterase activities; and liver glycogen and blood glucose levels were determined 5, 10, and 15 hours after intraperitoneal administration.
Results demonstrated an increase in cyclic AMP levels following both routes of atrazine administration; however, the increase was more pronounced after the intraperitoneal injection than after oral dosing. The increase in liver cyclic AMP was highest 4 hours after injection, and was followed one hour later by a three-fold increase in glycogen phosphorylase activity. Intraperitoneal administration of atrazine also caused a decrease in liver glycogen and a 50% increase in blood glucose content within 4 hours of dosing. However, there were no changes in adenylate cyclase and phosphodiesterase activities. The authors stated that from these results it is evident that atrazine alters the intermediary metabolism of the rat [Messner et al., 1979].
in vitro, mice
The effect of atrazine on the active transport of glucose was studied in an isolated intestine from an unspecified strain of mice. A suspension of 0.1 mM atrazine was introduced to a 4-cm section of small intestine removed from a six-week-old male mouse, and was incubated for 80 minutes at 30 degrees. The results showed that atrazine significantly inhibited glucose transport in the serosal fluid (P<0.05) and the gut (P<0.01) [Guthrie et al., 1974].
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