The effects of atrazine on the sexual maturation of female rats.

The mammalian hazard assessment of the herbicide atrazine (ATR) has focused on the induction of mammary tumors and accelerated reproductive aging of adult rats, and the relationship of these effects to the inhibition of leutinizing hormone (LH) release from the pituitary, an effect itself caused by inhibition of GnRH signaling by the adult rat hypothalamus. In earlier studies, Laws et al. (Toxicol. Sci., 58, 366-376, 2000) demonstrated a delay in female rat sexual maturation induced by ATR, effects that could equally have been caused by inhibition of hypothalamic GnRH release. The present studies were designed to compare the doses that interfere with GnRH signaling seen in previous studies in adult Sprague-Dawley (SD) rats (LH surge suppression) with doses that impair GnRH signaling in peripubertal rats, as indicated by delayed sexual maturation. The studies evaluated the effects of ATR treatment on the timing of uterine growth and vaginal opening (VO) in peripubertal female Wistar (Alderley Park, AP) and SD rats. Doses of 10, 30, and 100 mg/kg ATR were administered daily from postnatal day (pnd) 21 to up to pnd 46. Determinations of uterine weight were made at pnd 30, 33, 43 (AP), and 46 (SD) and the timing of VO was also assessed in the last two of these experiments. The centrally acting GnRH antagonist Antarelix (ANT) was used as a positive control agent as it has previously been shown to prevent uterine growth and to delay VO in peripubertal AP rats. Uterine growth and VO were completely prevented in AP rats exposed to ANT. Uterine growth was delayed at pnd 30 and 33 in AP rats exposed to 100 mg/kg ATR, but this growth inhibition had been overcome by pnd 43. VO was significantly delayed in AP rats for the 100 mg/kg ATR dose. By pnd 46, VO was significantly delayed in SD rats exposed to both 30 and 100 mg/kg ATR, but uterine weights were unaffected by that time (as for AP rats). It is concluded that the no-effect level for the effects of ATR on sexually immature rats (10 mg/kg in SD; 30 mg/kg AP) is approximately the same as reported previously by Laws et al. in peripubertal Wistar rats (25 mg/kg). However, the no-effect level in peripubertal female SD rats is nearly an order of magnitude greater than the no-observed effect level observed in female SD rats fed ATR for 6 months (1.8 mg/kg) where LH suppression was used as an indicator of effect on the pituitary/hypothalamic axis (USEPA, Atrazine-DACT Fourth Report of the Hazard Identification and Review Committee, April 5, 2002). These results support the conclusion that the pituitary/hypothalamic axis in peripubertal female SD rats is less sensitive than that in adult female SD rats.

New wheelie aid for wheelchairs: controlled trial of safety and efficacy.

OBJECTIVE: To test hypotheses that people learning to perform aided wheelies (AW) with a new self-deploying wheelie aid (WA) (1) are safer than those who use the conventional wheelie (CW), (2) are more successful at learning the skills, (3) learn more quickly, and (4) find such skills less difficult. DESIGN: Randomized, controlled study. SETTING: Wheelchair obstacle course. PARTICIPANTS: Forty-two subjects (12 wheelchair users, 30 able-bodied) randomly assigned to the CW (n = 23) or AW (n = 19) groups. INTERVENTIONS: We performed static tests on a WA-modified wheelchair occupied by a test dummy. We also attempted to teach each subject to perform a set of 14 wheelie-related skills. MAIN OUTCOME MEASURES: Visual analog scale (VAS) of safety, percentage of subjects able to learn the skills, the time required, and subjective difficulty scores (from 1 for "very easy" to 5 for "very difficult"). RESULTS: Up to 11.2 degrees of antitip-device stability was available without the WA extending beyond the rearmost aspect of the rear wheel in the resting position. For the CW and AW groups, the mean +/- standard deviation VAS safety scores were 43% +/- 27% and 98% +/- 2% (p <.0001), respectively; the overall success rates were 93% and 96% (p =.079), respectively; the mean times required to learn a skill (in 5-min increments) were 1.56 +/- 1.08 minutes and.72 +/-.35 minutes (p =.002), respectively; and the mean difficulty scores were 2.94 +/-.38 and 2.23 +/-.34 (p <.0001), respectively. CONCLUSIONS: The new WA provides stability and wheelie-like function without interfering with maneuverability. Although both groups were equally successful, learning to perform AWs is safer, faster, and less difficult than learning CWs.

Atrazine effects on testosterone levels and androgen-dependent reproductive organs in peripubertal male rats.

Previous studies have reported that atrazine, a widely used herbicide that selectively inhibits photosynthesis in broadleaf and grassy weeds, has adverse effects on reproductive function in the male, suggesting a direct effect of atrazine on the hypothalamicpituitary-testicular axis. As yet, however, no studies have critically examined the doses of atrazine that elicit such effects, and few have focused on the mechanism by which atrazine acts. Herein we report a dose-response study of the effects of atrazine ingestion on reproductive function in male Sprague-Dawley rats during a critical developmental period, the peripubertal period. Atrazine was administered by gavage to rats from day 22 to day 47 of age, at doses of 1-200 mg/kg body weight per day. Atrazine administration of up to 50 mg/kg per day had no effect on any of the measured variables. Serum testosterone concentration was reduced by atrazine at doses of 100 and 200 mg/kg per day, as were seminal vesicle and ventral prostate weights. Intratesticular testosterone concentration was reduced in parallel with serum testosterone, suggesting that the reductions in serum testosterone resulted from reduced testosterone production by Leydig cells or from changes in testosterone metabolism within the testis, or both. Serum luteinizing hormone (LH) concentration was reduced despite the reduced serum testosterone, suggesting an effect on the hypothalamus, the pituitary gland, or both. At the termination of the study, the average body weight of rats receiving atrazine at 100 mg/kg per day was found to be reduced by approximately 9%. This suggested the possibility that the effects of atrazine on the reproductive tract may not be direct, but rather, the noted deficits of the male reproductive tract resulted from reduced food intake by the treated rats. We tested this by feeding control (vehicle-gavaged) rats amounts of food equivalent to that consumed by the atrazine-fed rats, and then assessing reproductive tract endpoints. Even mild food restriction resulted in reductions in serum testosterone concentration, in the weights of androgen-dependent organs, and in serum LH concentration; the same deficits that were seen in atrazine-gavaged rats. Indeed, the effects of atrazine on the male reproductive tract seen in rats receiving atrazine at greater than 50 mg/kg per day could not be distinguished from the effects of reduced food consumption. These results suggest that caution must be exercised before concluding that atrazine (or any potentially toxic chemical) has direct, detrimental effects.

A risk characterization for atrazine: oncogenicity profile.

An extensive safety database has been developed for the chlorotriazine herbicide, atrazine. The results from five oncogenicity studies conducted in the Sprague-Dawley rat, two studies in the Fischer 344 rat, and two studies in the CD-1 mouse were reviewed. No increase in the incidence of tumors of any type was observed in male or female Fischer 344 rats, male or female CD-1 mice, or male Sprague-Dawley rats fed atrazine at a maximum tolerated level in their diet for 24 mo. Female Sprague-Dawley rats fed atrazine at levels of 400, 500, and 1000 ppm developed mammary tumors earlier than did the control group. The incidence of female Sprague-Dawley rats with mammary tumors after 24 mo of treatment was statistically increased at feeding levels of > or = 70 ppm in 1 study and at 400 ppm in a second study, whereas there were no significant differences between the treated and the control group in 3 other studies. No increase in tumors of any type was observed in ovariectomized female Sprague-Dawley rats after 24 mo of atrazine treatment at the highest level tested, 400 ppm. Therefore, the mammary tumor response in female Sprague-Dawley rats following the administration of high levels of atrazine appears to be due to an acceleration of the normal reproductive aging process resulting in increased exposure to endogenous estrogen and prolactin. The Sprague-Dawley rat differs from the Fischer 344 rat, the CD-1 mouse, and humans in the endocrine control mechanisms affecting reproductive senescence and the development of the mammary tumors during aging. These data indicate that the carcinogenic effect of high doses of atrazine observed in the female Sprague-Dawley is a strain-, sex-, and tissue-specific response that does not have biological relevance to humans.

Comparison of Lp(a) concentrations and some potential effects in hemodialysis, CAPD, transplantation, and control groups, and review of the literature.

Apolipoprotein (a)-Lp(a)-is reported to be an independent risk factor for coronary artery disease and for hemodialysis (HD) access occlusion. Homology with plasminogen may predispose to thrombosis. High concentrations usually have been reported in patients on HD and on continuous ambulatory peritoneal dialysis (CAPD), but near-normal values in many kidney transplants (TP). We used Pharmacia immunoradiometric assay in 52 patients on HD, 58 on CAPD, 94 after TP, and 56 controls. The Lp(a) mean levels for CAPD, HD, TP, and control groups were 738, 647, 348, and 368 U/l and the medians were 542, 537, 96 and 143 U/l, respectively. The means and medians for CAPD and HD were significantly greater than those for TP and controls (p < 0.003 for means and < 0.005 for medians). We found no significant difference between: (1) Lp(a) means or medians comparing HD and CAPD or TP and controls; (2) Lp(a) means for the 33 patients with insulin-dependent diabetes mellitus and the 171 without; (3) number of occlusions of HD fistulae or grafts in patients with high Lp(a) values and without; (4) mean Lp(a) for CAPD patients on gemfibrozil and also for TP patients on 3-hydroxy-methylglutaryl coenzyme 1 reductase inhibitors, or diet alone, before and after treatment, and (5) mean Lp(a) values for HD and CAPD patients with and without myocardial infarction. Lp(a) did not correlate significantly with fractional shortening or left ventricular end systolic or diastolic diameter by echocardiogram or with ejection fraction. For TP patients, Lp(a) and serum creatinine correlated (p = 0.004), and mean Lp(a) for 71 TP on ciclosporin A exceeded that for the other 23 patients (p < 0.03). Lp(a) fell in 13 of 14 patients after TP (mean fall 77%). The dominant Apo(a) isoform in 10 of 13 patients on CAPD or HD with high Lp(a) values was the equivalent of S2 (Utermann). Lp(a) in HD or CAPD is often elevated and regulated by both genetic and renal failure factors, but falls after TP with return of renal function and mainly genetic regulation. Lp(a) was not a risk factor for coronary artery disease in HD or CAPD patients and did not fall significantly with two drugs or diet.

Factors affecting mammary tumor incidence in chlorotriazine-treated female rats: hormonal properties, dosage, and animal strain.

Chlorotriazines are widely used in agriculture as broadleaf herbicides. The compounds specifically inhibit photosynthesis, and, as such, display little interaction with animal systems. However, a 24-month feeding study with atrazine (ATR) revealed a significant dose-related increase of mammary tumors in female Sprague-Dawley (SD) rats. Because numerous studies indicated that ATR had a low mutagenic and oncogenic potential, it was decided to test a hypothesis that the herbicide possessed endocrine activity. Among tests for estrogenic action, oral dosing of ATR up to 300 mg/kg did not stimulate uterine weight of ovariectomized rats. However, ATR administration did reduce estrogen-stimulated uterine weight gain. Further evidence of inhibition came from measures of [3H]-thymidine incorporation into uterine DNA of ATR-treated immature rats. Again, no intrinsic estrogenic activity was observed up to a 300-mg/kg dose. In vitro, ATR competed poorly against estradiol binding to cytosolic receptors, with an approximate IC50 of 10(-5) M. Atrazine administration to SD and Fischer-344 (F-344) rats for 12 months, up to 400 ppm in food, was correlated with significant alterations of estrous cycling activity; but there was a divergent strain response. SD rats showed an increased number of days in vaginal estrus, increased plasma estradiol, and decreased plasma progesterone by 9 to 12 months of treatment. F-344 rats did not demonstrate treatment-related affects. A study of ultrastructure in the hypothalamic arcuate nucleus of female SD rats that were fed diaminochlorotriazine (DACT), an ATR metabolite, suggested that age-associated glial pathology was enhanced by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothesis for mammary tumorigenesis in Sprague-Dawley rats exposed to certain triazine herbicides.

The symmetrical triazine herbicides have been used for the preemergence control of broadleaf weeds for nearly three decades. Recently, certain members of this class, primarily the chlorotriazines (substituted in the 2 position), have been shown to evoke an increased incidence of mammary tumors in female Sprague-Dawley rats. This response was noted when these chemicals were administered in the diet for 2 yr, and most often at dietary feeding levels at or above the maximum tolerated dose (MTD). At levels exceeding the MTD the health of these animals was compromised, as manifested by toxicity-related reduced survival that was not associated with the occurrence of mammary tumors. Mammary tumors in rats frequently occur as a result of the influence of endogenous estradiol and prolactin. Those hormones, as well as progesterone, growth-stimulating, luteinizing, and follicle-stimulating hormones, were measured after 24 mo of dietary administration of the chlorotriazine, simazine. The plasma hormone pattern seen in aged female Sprague-Dawley rats administered 1000 ppm simazine in the diet for 24 mo resembled that noted for aged female controls, except that the difference was more pronounced in the simazine-treated group. These results suggest that prolonged exposure of Sprague-Dawley females to excessive levels of triazines affects the neuroendocrine system, which in turn alters the pathology of the mammary gland. These changes are comparable to those that occur naturally as the rat ages. Changes in neuroendocrine control could result in the expression of an earlier onset and/or an increased incidence of mammary tumors, which already occur at a high spontaneous rate in aging Sprague-Dawley female rats.

Short-term effects of chlorotriazines on estrus in female Sprague-Dawley and Fischer 344 rats.

Atrazine or simazine (s-chlorotriazines) was administered by gavage daily for 2 wk to female Sprague-Dawley and Fischer 344 rats at oral doses of 100 or 300 mg/kg to evaluate effects on body, ovary, uterus, and adrenal weights, estrous cycle stages, vaginal cytology, and plasma hormone (estradiol, progesterone, prolactin, and corticosterone) levels. Significant reductions in body weights of both Sprague-Dawley and Fischer 344 female rats at both dose levels were accompanied by a significant reduction in ovarian and uterine weights, and a decrease in circulating estradiol levels. The magnitudes of the effects were less in Fischer 344 rats than in Sprague-Dawley rats, and the effects of simazine were less pronounced than those of atrazine at the same dose. A maximum tolerated dose (MTD: > or = 10% body weight reduction) was estimated to be 100 mg/kg for atrazine and 300 mg/kg for simazine for both stains. The Sprague-Dawley female rats exhibited a treatment-related lengthening of the estrous cycle and an increased number of days characterized by cornified epithelial cells. This resulted in a greater percent of the cycle days spent in estrus and reduction in the percent of the cycle days spent in diestrus. Atrazine-dosed Fischer 344 females also exhibited a significant trend toward cycle lengthening, but this was due to reduction in the percent of cycle spent in estrus and a concomitant increase in diestrual days. These findings suggest that treatment with doses of triazine at or above the MTD may result in prolonged exposure to endogenous estrogen in the Sprague-Dawley but not the Fischer 344 rat. These changes may account for the observed earlier onset and/or increased incidence of mammary tumors in chlorotriazine-treated female Sprague-Dawley rats. This strain of rat is already known to be prone to a substantial development of mammary tumors with advancing age, while the Fischer 344 strain is not as likely to exhibit this response.

Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fischer 344 rats.

The chronic effects of dietary administration of atrazine at levels as high as 400 ppm on selected endocrine and tumor profiles were evaluated in Fischer 344 and Sprague-Dawley female rats. The study showed that lifetime dietary administration of atrazine at a maximum tolerated dose (MTD) to Sprague-Dawley female rats caused (1) lengthening of the estrous cycle, (2) increased number of days in estrus or under the influence of exposure to estrogen, (3) earlier onset of galactocele formation, and (4) earlier onset of mammary and pituitary tumor formation but not an increased incidence of mammary and pituitary tumors when compared to concurrent control rats. Fischer 344 female rats fed atrazine at an MTD exhibited slightly lengthened estrous cycles, but no effects were observed on estradiol or progesterone levels, or on the onset or incidence of mammary tumors. These results support a hypothesis that high-dose atrazine administration in Sprague-Dawley females is related to an acceleration of age-related endocrine changes leading to an earlier onset and/or increased incidence of mammary tumors. This endocrine-mediated response, which appears to be unique to the Sprague-Dawley female rat, occurs only at or above a threshold dose (the MTD) that interferes with normal estrous cycling, promoting prolonged exposure to endogenous estrogen.

Possible antiestrogenic properties of chloro-s-triazines in rat uterus.

Several published reports have indicated that certain chloro-s-triazine herbicides may alter endocrine function in rats, possibly by androgen receptor binding. In direct tests of estrogenic bioactivity, oral doses of up to 300 mg/kg/d of atrazine, simazine, or the common metabolite diaminochlorotriazine (DACT) did not significantly increase uterine weight of ovariectomized Sprague-Dawley female rats. The highest dose, which was approximately 10% of the LD50 for these compounds, did cause body weight loss. When administered concomitantly with sc injections of estradiol (2 micrograms/kg), 300 mg/kg of orally administered chlorotriazines significantly reduced uterine weight in comparison to animals given estrogen alone. Neither atrazine, simazine, nor DACT, at oral doses up to 300 mg/kg/d, stimulated incorporation of [3H]thymidine into uterine DNA of immature Sprague-Dawley female rats. However, oral treatment at doses of 50 mg/kg and higher significantly reduced thymidine incorporation into uterine DNA extracted from immature rats given a single injection of 0.15 microgram estradiol. Oral doses of 300 mg/kg of atrazine, simazine, or DACT significantly reduced expression of progesterone receptor binding in cytosol fractions prepared from uteri of ovariectomized rats injected sc with 1 microgram estradiol; 50 mg/kg triazine was not effective in this case. Uterine progesterone receptor levels were not stimulated in rats given oral doses up to 300 mg/kg of these triazines without estradiol injections. These results suggest that atrazine, simazine, and DACT possess no intrinsic estrogenic activity but that they are capable of weak inhibition of estrogen-stimulated responses in the rat uterus. This inhibition may play a role in the previously observed disruptive actions of chlorotriazines on reproductive endocrine function of female rats.

Chloro-s-triazine antagonism of estrogen action: limited interaction with estrogen receptor binding.

In an accompanying article (see pp. 183-196), it was reported that administration of very high doses of the chlorotriazine herbicides atrazine, simazine, and diaminochlorotriazine (DACT), a common metabolite, expressed antiestrogenic activity in uteri of female Sprague-Dawley rats without expressing intrinsic estrogenic activity. In the present article, studies of chlorotriazine interaction with rat uterine estrogen receptors (ER) are reported. Under equilibrium conditions, none of the triazine compounds showed an ability to compete against binding of radiolabeled estradiol to ER. A weak competition was evident only if cytosols were preincubated with triazines at 25 degrees C prior to introduction of tracer. Competition was very weak, with kl estimates of 10-100 microM. A limited Scatchard analysis suggested a competitive type of inhibition. Sucrose gradient analysis of cytosol incubations showed that triazine interaction with the 4S isoform of ER may be greater than with the 8S form. When administered to ovariectomized rats for 2 d at 300 mg/kg/d, atrazine, simazine, or DACT all reduced uterine ER binding capacity by approximately 30%. Results from the receptor binding studies indicated that triazine competition against ER binding occurred to a much lesser degree than inhibition of estrogen-mediated responses reported in accompanying articles. This suggests that the complete responses to triazines may include inhibition of events other than or in addition to ER binding of estrogen.

A subchronic inhalation toxicity study of a Matacil formulation in the albino rat.

The subchronic toxicity of a new formulation of Matacil (aminocarb) was assessed by exposing male and female Sprague-Dawley rats via a nose-only technique to a respirable (2.0- to 4.1-microns diameter) aerosol at chamber concentrations of 22.5, 45, and 90 micrograms of insecticide/liter of air for 2 hr/day for 30 consecutive days. Control groups were exposed to a vehicle aerosol or to room air. Randomly selected rats of each group were bled after 8, 15, and 30 days of treatment, and after a 30-day recovery period. Routine clinical laboratory investigations (hematology, blood chemistry, and urinalysis) were conducted during treatment. Other parameters measured included body weight, feed intake, plasma, red blood cell count, brain cholinesterase activity, and hepatic and renal carboxylesterase activities. Organ weights were recorded at necropsy and routine histopathological evaluation was performed. Mild muscle tremors were observed occasionally in the intermediate- and high-dose groups. Treated females, but not males, demonstrated a dose-dependent inhibition of cholinesterase activity, though within treatment groups, there were no differences associated with the number of days of treatment. Enzyme values had returned to baseline levels by 30 days post-treatment. Hepatic carboxylesterase activity was significantly reduced only in male rats at the highest dose. Lung weights were increased in vehicle and Matacil-treated groups. Histological studies indicated that these changes were a nonspecific tissue response to a heavy burden of an oil-based irritant, which was partially resolved by 30 days post-treatment. The results showed that, at the concentrations tested, the formulation produced little or no acute symptoms and minimal long-term sequellae.

Six month inhalation studies of pirbuterol acetate aerosol in the beagle dog and squirrel monkey.

Groups of Beagle dogs and Squirrel monkeys were exposed to aerosols of pirbuterol acetate, a new bronchodilator at doses of 0, 200, 400 and 800 micrograms of pirbuterol/kg body weight daily for 6 months. Each group consisted of 4 dogs or 6 monkeys per sex. Dogs were exposed by face mask and monkeys were exposed by head only in a manifold. Plasma drug concentrations indicated that expected levels of drug exposure were achieved in both species. No significant alterations were revealed in physical appearance and body weights, hematological and blood biochemical analyses, urinalyses, ophthalmoscopy, assessment of cardiovascular status, pulmonary function or gross and histopathology. Examination of the respiratory tract showed no morphological changes that could be attributed to pirbuterol acetate inhalation.

Some preliminary observations on the effects of kola nut on the cardiovascular system.

The prevalence of kola nut chewing and the effects attributed to it are briefly reviewed. Except for a generalisation that its effects are probably due to its caffeine content, no attempts have been made to quantify its specific actions. Preliminary observations on the cardiovascular system of the cat show that aqueous extracts of the nut evoke a dose dependent differential response - the 8 - 9% hypertensive effect of 0.01 ug - 1mg/ml doses contrasting with the 18--71% hypotensive response of 10--1000mgs/ml doses. Adrenergic receptor blockade showed no significant alteration of the response. With larger doses a bradycardia developed and cardiac arrythmias were observed as a terminal event. These results are compatible with the action of xanthines on the heart and peripheral vasculature. The possible role of the habitually chewed kola nut in the pathophysiology of cardiovascular disease and its implications have been briefly discussed.

Multi-laboratory comparison of three heparin-Mn2+ precipitation procedures for estimating cholesterol in high-density lipoprotein.

Plasma high-density lipoprotein is commonly estimated by measuring the cholesterol remaining in plasma supernatant solutions after other lipoproteins, which contain apolipoprotein B, are precipitated with heparin and Mn2+. The method (method I) now in use by the Lipid Research Clinics, in which Mn2+ is at 46 mmol/liter final concentration, is reasonably accurate, but precipitation and sedimentation of lipoproteins other than high-density lipoproteins is often incomplete. We evaluated two modifications of method I. In method II, the Mn2+ concentration was doubled; the second modification (method III) included the increased Mn2+ concentration in a combined heparin Mn2+ reagent, decreased sample volume (2 ml), and a shorter incubation time (10 min at room temperature). The percentages of samples with turbid supernates (i.e., incomplete sedimentation) by methods I, II, and III were 9, 3, and 2%, respectively. Among non-turbid supernates, the percentages of samples containing measurable apolipoprotein B (incomplete precipitation) were 79, 19, and 16%, respectively. We conclude that method III is the most convenient and accurate of the three procedures.