CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, North Carolina 27709
Received January 17, 2002; accepted February 20, 2002
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ABSTRACT |
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Key Words: fenitrothion; reproductive development; androgen receptor antagonist; in utero exposure; anogenital distance; nipple retention.
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INTRODUCTION |
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In response to concerns about risk to children's health from environmental exposures, the U.S. Food Quality Protection Act (FQPA) was passed in 1996 and the Safe Drinking Water Act amended (Kimmel and Makris, 2001). These changes required the U.S. Environmental Protection Agency (U.S. EPA) to develop a screening program to identify compounds with potential endocrine activity. The U.S. EPA created the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) in 1996 to provide advice on designing a screening and testing program capable of detecting endocrine-active compounds. EDSTAC recommended a framework that includes a screening battery (Tier 1) to detect (anti)estrogenic, (anti)androgenic, and antithyroid activities using in vitro assays (e.g., estrogen and androgen receptor binding-reporter gene assays) and short-term in vivo assays of pharmacological activity (e.g., rodent Hershberger and uterotrophic assays) (Gray, 1998
; U.S. EPA, 1998
). Chemicals testing positive in Tier 1 would be labeled as potential endocrine disrupters and subjected to more extensive testing (Tier 2) to determine whether the compound exhibits endocrine-mediated adverse effects in vivo and to characterize and quantify those effects during the most sensitive stages of development. The EDSTAC recommendations have instigated an intensive effort by researchers to determine which in vitro and in vivo assays will prove most useful.
Organophosphate insecticides are widely used in agriculture to control pests in the environment, and there is considerable human exposure to this class of compounds. Moreover, children's health may be at risk from exposure to these types of chemicals (Eskenazi et al., 1999; Landrigan et al., 1999
; Lu et al., 2001
). Organophosphate insecticides are well-characterized neurotoxins; they inhibit cholinesterase activity and induce cholinergic stress (Pope, 1999
). Recently, the organophosphate insecticide fenitrothion [0,0-dimethyl-O-(4-nitro-m-tolyl) phosphorothioate] was found to act as an antiandrogen using in vitro and in vivo screening assays (Tamura et al., 2001
). In an in vitro assay, fenitrothion competitively antagonized human androgen receptor activity in transfected cells (KB value of 2.18 x 10-8M) (Tamura et al., 2001
). This showed that fenitrothion is comparable to flutamide in potency (KB value of 1.07 x 10-8M) (Maness et al., 1998
) and 30-fold more potent than linuron (KB value of 75.8 x 10-8M) (McIntyre et al., 2000
). In addition, fenitrothion treatment decreased the weights of the ventral prostate, seminal vesicles, and LABC muscle in a Hershberger assay using castrated adult male rats treated with 15 and 30 mg/kg/day fenitrothion and 50 µg/day testosterone propionate (Tamura et al., 2001
).
Because androgens are required during late gestation for sexual differentiation and because fenitrothion antagonizes the androgen receptor in vitro and in vivo (Tamura et al., 2001), we hypothesized that exposure to fenitrothion during this critical period in utero impairs androgen-mediated development, resulting in abnormalities of the reproductive tract in male offspring. The purpose of this study was to determine whether in utero exposure to fenitrothion during gestation day (GD) 12 to 21 induces dose-responsive alterations in androgen-dependent developmental end points such as AGD, nipple retention, preputial separation, and testicular and epididymal development.
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MATERIALS AND METHODS |
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Treatment.
The study was divided into two blocks. In the first block (27 presumed pregnant dams), six dams were allocated to the control group (0 mg/kg/day), and seven dams were each allocated to the groups receiving 5, 10, or 15 mg/kg/day fenitrothion (>98% purity, Chem Service Inc., West Chester, PA). A second block of dams (17 presumed pregnant dams) was started on the study once the offspring of the first block had been weaned, as maternal toxicity was not observed in the dams receiving 15 mg fenitrothion/kg/day. Five dams were allocated to the control group (0 mg/kg/day) and six dams each to the groups receiving 20 and 25 mg/kg/day fenitrothion. The dams were gavaged daily (between 8:00 and 9:00 A.M.) from GD 12 to 21 with either corn oil (Sigma Chemical Company, St. Louis, MO) or fenitrothion in corn oil at a volume of 1 ml/kg/day. The dose levels of fenitrothion selected for this study were chosen to demonstrate maternal toxicity at the highest dose level tested and to characterize potential dose-response relationships. The doses selected were based on a summary document published by the U.S. EPA (1997) in which the no observed adverse effect level (NOAEL) for maternal toxicity was 8 mg/kg/day and the lowest observed adverse effect level (LOAEL) was 25 mg/kg/day in a rat developmental toxicity study. Dams were examined twice daily for clinical signs of toxicity. Dam body weights were recorded daily during dosing and weekly during lactation. Dam food consumption was monitored weekly throughout dosing and lactation.
Abbreviated functional observational battery.
An abbreviated functional observational battery (FOB) was performed on GD 12 on all dams in the study beginning 2 h after dosing, and additionally on GD 19 on the block 2 dams beginning 6 h after dosing. The order of the abbreviated FOB testing was randomized, and the FOB was performed by an experienced investigator who was unaware of the exposure group of each animal, according to methods described previously (Dorman et al., 2000; Moser et al., 1988
). Each dam was observed in its home cage, on removal from the cage while being held, as it moved freely about an open field, and during manipulative tests. Each animal was evaluated for posture, tremors, spasms, convulsions, palpebral closure, handling reactivity, and muscle tone. The condition of the animal was also noted and included piloerection, lacrimation, salivation, fur appearance, facial crust, skin temperature and color, and breathing pattern. The animal was then assessed during a 1-min observation period in an open field 75 x 38 x 20 cm with clean techboard on the floor. Assessments included arousal, activity, ataxia, gait, body position, excessive vocalization, tremors, spasms, seizures, and unusual behaviors. The observation period was followed by manipulative procedures that included an assessment of visual approach response, acoustic response, tail-pinch response, visual placing, and surface-righting reflex.
Androgen-dependent reproductive end points.
Following delivery of the entire litter, the live pups were sexed, counted, and examined for clinical signs of toxicity; mortality was also recorded. The length of the perineum from the base of the sex papilla to the proximal end of the anal opening (AGD) of both male and female pups was measured using a dissecting microscope with an eyepiece reticle (accuracy, 0.05 mm). A single investigator unaware of the animal exposure group performed all the measurements. Individual pup weights and pup litter weights (by sex and litter) were collected on PND 1. Pup litter weights were also collected on PND 4, 7, 14, and 21. At weaning, all the offspring were ear-tagged, and individual body weights were recorded weekly.
On PND 13, male pups were inspected for the presence of areolae. A single investigator unaware of the exposure group of the animal recorded the number and location of the areolae. An animal was considered a responder if it displayed more than one retained areola. Vaginal opening was monitored daily from PND 28 until each female acquired this developmental landmark or until PND 48, whichever came first. Males were examined for preputial separation from PND 38 until the prepuce could be completely retracted from the glans penis and was complete by PND 55. During this period, animals were also inspected for cryptorchidism and hypospadias.
Necropsies.
Pups were weaned on PND 21, and dams were euthanized by CO2 asphyxiation. Maternal body and organ weights (liver, kidneys, and uterus) and number of implantation sites were recorded. The sexually mature female offspring (6065 days old) were euthanized by CO2 asphyxiation, and body and organ weights (liver, kidneys, adrenals, brain, ovaries, and uterus) were recorded. Male offspring were euthanized by decapitation at sexual maturity (96105 days old), external genitalia were visually inspected, AGD was measured, and the presence of nipples was noted following shaving of the thorax and abdomen. All animals were subjected to a macroscopic internal examination before organs were prosected and weighed. Body and organ weights (liver, kidneys, adrenals, brain, testes, epididymides, vasa deferentia, prostate, seminal vesicles with coagulating glands and seminal fluid, and LABC muscle) were collected. Tissues were fixed in either Bouin's fixative (testes and epididymides) or 10% neutral buffered formalin, paraffin-embedded, sectioned (5 µm), processed, and stained with hematoxylin and eosin. Histopathology was performed on the following male tissues: testes, epididymides, prostate, liver, kidney, and adrenal glands.
Statistical analysis.
The litter was the experimental unit, and statistical analyses were conducted using JMP (version 4.0, SAS Institute, Cary, NC). Normality (Shapiro-Wilk) and homogenicity of variances (Bartlett) were tested prior to data analysis. Because the proportion of pups born alive, pups surviving to weaning, and sex ratio was not normally distributed, an arcsine transformation was conducted prior to analysis. Pups were nested by dam to yield litter means. Either analysis of variance (ANOVA) or analysis of covariance (ANCOVA) was used to test for significance of treatment effects; covariants are listed in the table legends. If the p value for treatment effects was less than 0.05, contrasts of least square means were used to assess the significance of treatment differences. Analysis was performed to assess significant block effects on the control data. Where there was no significant block effect, the data from the individual blocks was combined for statistical analysis. If a difference between the two sets of control data was observed, the data for the individual blocks are shown in the results tables. The data presented are means or least square means ± SE.
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RESULTS |
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Measurement of AGD and nipple counts in the adult male offspring at necropsy indicated that the decrease in AGD observed at PND 1 and the increase in the number of areolae at PND 13 were transient effects. At PND 100, AGD was similar in all the groups (Fig. 1), and none of the male animals displayed nipples. The adrenal glands were the only tissues to display a significant increase in weight (13%), and this effect was only apparent in the 20 mg/kg/day exposure group (Table 3
). This effect was not dose responsive, and no pathology was observed histologically. Body weights and all other organ weights were unaltered in the male offspring.
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DISCUSSION |
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The measurement of AGD and areola retention in early postnatal life are considered to be relatively sensitive end points of altered androgen action during development (Clark et al., 1990; Gray et al., 1999a
,b
; Hellwig et al., 2000
; McIntyre et al., 2000
, 2001a
, b
; Mylchreest et al., 1999
, 2000
; Ostby et al., 1999
; Parks et al., 2000
). Late gestational exposure to 20 and 25 mg/kg/day fenitrothion decreased AGD on PND 1 in the male offspring by 8 and 16%, respectively. The size of the decrease is comparable to that seen following exposure in utero during the same period of gestation to 50 mg/kg/day linuron (McIntyre et al., 2000
, 2001b
) and to 250 mg/kg/day DBP (Mylchreest et al., 1999
, 2000
). Further confirmation of the ability of fenitrothion to disrupt androgen-mediated development in rats was provided by the increase in number of retained areolae in male offspring on PND 13 from approximately one in the control group to four in the 25 mg/kg/day fenitrothion-exposed group. The effect of fenitrothion on areolae retention was similar to that seen following in utero exposure to 50 mg linuron kg/day, which resulted in 34 areolae/pup compared with less than one areola in the control group (McIntyre et al., 2000
, 2001b
).
Measurement of AGD at PND 100 revealed that the decreases observed in PND 1 males were transient and that the length of the perineum was the same in the fenitrothion-exposed male offspring as that in the control animals. Similarly, no retained nipples were apparent in the adult male rats. In contrast, linuron exposure resulted in permanent effects in both of these end points, even though the magnitude of the change was decreased in the older animals (McIntyre et al., 2001b). Transient effects have been described in male offspring exposed to low dose levels of finasteride (Clark et al., 1990
) and vinclozolin (Hellwig et al., 2000
). Clark et al. (1990) showed that AGD was permanently decreased following exposure to 100 mg/kg/day finasteride but fully recovered at PND 140 after exposure to 0.1 mg finasteride/kg/day. Hellwig et al. (2000) found that exposure to 200 mg/kg/day vinclozolin from GD 14 through PND 3 induced a significant increase in nipple retention at PND 13 (litter response rate 100%) that was still apparent on PND 180. Exposure to 12 mg/kg/day vinclozolin increased the numbers of areolae/nipples at PND 13, but most animals did not exhibit nipples on PND 180 (Hellwig et al., 2000
). The changes in AGD and retention of areolae are indicative that androgen status of the fetus was impaired by exposure to fenitrothion, but the fact that the changes are transient means that they would not normally be considered adverse effects.
Obvious signs of cholinergic stress were observed in the animals administered the two highest doses of fenitrothion (20 and 25 mg/kg/day); these signs included muscle tremors and decreases in body weight gains. Similarly, dosing of pregnant rats with 25 mg/kg/day daily or 30 mg/kg fenitrothion every other day during GD 6 through 15 resulted in maternal toxicity and mortality with the higher dose level of fenitrothion (Berlinska and Sitarek, 1997; U.S. EPA, 1997
). The LOAEL for maternal toxicity in a standard developmental toxicity study was found to be 25 mg/kg/day fenitrothion; based on the results of the present study, the LOAEL could be revised to 20 mg/kg/day. No adverse effects were apparent during late gestation when the animals were treated with 15 mg/kg/day. Maternal cholinesterase activity is the most sensitive indicator of organophosphate exposure, and significant inhibition of cholinesterase activity can be observed in the absence of any clinical findings (Astroff and Young, 1998
). Maternal cholinesterase activity was probably inhibited at doses lower than 20 mg/kg/day fenitrothion, as treatment of adult male rats with 15 mg/kg/day for 7 days was sufficient to reduce brain, but not plasma, cholinesterase activity in a Hershberger assay (Tamura et al., 2001
).
Fenitrothion exposure also induced fetotoxicity, as evidenced by an increased incidence of fetal death. The number of pups born alive following late gestational exposure to 20 and 25 mg fenitrothion/kg/day was decreased, even though the number of implantation sites in these two exposed groups was similar to that found in the control litters. This implies that fenitrothion induced postimplantation losses and decreased fetal viability. These effects were apparent only at doses that induced maternal toxicity. Previous investigators have observed similar effects. For example, an increased frequency of early resorptions and postimplantation losses was observed in rats following exposure to 30 mg/kg fenitrothion from GD 6 to 15 (Berlinska and Sitarek, 1997). Significant mortality (around 17%) was observed in pups up to PND 16 following gestational exposure to 5, 10, and 15 mg/kg/day fenitrothion (Lehotzky et al., 1989
). In addition, a two-generation reproduction study reported decreases in fertility in the F0 generation, numbers of implantation sites, and viability following dietary exposure to 120 ppm fenitrothion (U.S. EPA, 1997
). In contrast, a standard developmental toxicity study in rats for fenitrothion found no evidence of increased incidence of pup death, postimplantation loss, or teratogenicity following treatment of pregnant Sprague-Dawley rats with 25 mg/kg/day fenitrothion during GD 6 to 15 (U.S. EPA, 1997
). Currently, the LOAEL for developmental toxicity in rats has been set at 25 mg/kg/day based on an increased incidence of fetuses and litters with supernumerary ribs (U.S. EPA, 1997
). The present data suggest that adverse effects on development can be induced by exposure to 20 mg fenitrothion/kg/day during GD 12 to 21.
Fenitrothion provides a useful illustration of the efficacy of the EDSTAC-proposed Tier 1 and Tier 2 screening and testing methodologies. In the present study, we were able to confirm the antiandrogenic activity of fenitrothion identified using the Tier 1 screens and to demonstrate that fenitrothion is antiandrogenic during pregnancy. Fenitrothion is a potent inhibitor of acetylcholinesterase and was found to induce maternal toxicity (cholinergic stress and decreased body weight gain) and fetal mortality at the same doses as those shown to affect AGD and areolae retention, albeit the effects on AGD and areolae retention were transient in nature. Within the confines of this study, any potential risk assessment of exposure to fenitrothion based on its neurotoxicity effects will probably be protective of its reproductive effects. The doses used in this study are relevant for risk assessment purposes. Dietary exposure to fenitrothion is considered minimal, and there is a lack of accurate exposure data for occupational use of fenitrothion. Daily dermal exposures have been estimated to range from 0.45 to 2.65 mg/kg/day (U.S. EPA, 1994). The LOAEL for transient effects on AGD and areolae retention is 25 mg/kg/day, which is approximately 9-fold higher than the maximal dermal exposure reported for human occupational use of fenitrothion.
Extrapolation of the results of the in vitro and in vivo screening assays implied that fenitrothion would be a more potent antiandrogen than linuron but less active than flutamide in vivo. The weaker than expected antiandrogenic activity of fenitrothion in the present study in comparison with the Hershberger assay is most likely explained by its metabolism by the pregnant rat. In the Hershberger assay, fenitrothion is administered to adult male castrate rats, which do not possess a functioning hypothalamo-pituitary axis. In intact adult rats, fenitrothion is absorbed rapidly and excreted within 48 h, with around 90% and 5% of the excreted dose eliminated in urine and feces, respectively (U.S. EPA, 1994). Fenitrothion undergoes oxidative desulfuration in the liver. This biotransformation by the cytochrome P450 system results in the formation of a fenitrothion oxon that is a potent acetyl cholinesterase inhibitor (Levi et al., 1988
; Sultatos, 1994
), but has no antiandrogenic activity in an in vitro assay containing serum (H. Tamura, unpublished data).
The majority of environmental antiandrogens that have so far been evaluated for effects during in utero development are either metabolized to more potent antiandrogens, for instance, flutamide to hydroxyflutamide (Neri, 1989), or to a metabolite that maintains its antiandrogenic activity, albeit weaker than the parent compound as for linuron (Cook et al., 1993
). Clearly, the doses of fenitrothion in the Hershberger assay are sufficient to induce effects on androgen-dependent organ weights. However, for fenitrothion to alter male reproductive development in utero, fenitrothion must cross the placenta to gain access to the fetuses. In light of our present results, it seems likely that the transient effects of fenitrothion on AGD and areolae retention reflect a much lower effective dose delivered to the fetus due to maternal metabolism of fenitrothion to less active metabolites.
The results of the present study highlight the need for caution when evaluating the results from in vitro and in vivo screening assays for endocrine disrupters. These assays will not necessarily provide an accurate reflection of the potency of the chemical in vivo, because in vitro assays of receptor binding or receptor-mediated transcription incorporate no or very limited metabolic capability. The Hershberger assay is an extremely useful pharmacological screen for detecting antiandrogenic activity if performed correctly; however, it is not designed to characterize dose-response relationships for antiandrogenic effects. Information on the metabolism and tissue dosimetry of the agent in an intact animal is clearly an important contributor toward understanding potential effects in vivo. These parameters should be included in the analysis of potential risk of endocrine-active chemicals.
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ACKNOWLEDGMENTS |
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NOTES |
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