Effects of in Utero Exposure to the Organophosphate Insecticide Fenitrothion on Androgen-Dependent Reproductive Development in the Crl:CD(SD)BR Rat

Katie J. Turner,1, Norman J. Barlow, Melanie F. Struve, Duncan G. Wallace, Kevin W. Gaido, David C. Dorman and Paul M. D. Foster

CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, North Carolina 27709

Received January 17, 2002; accepted February 20, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fenitrothion [0,0-dimethyl-O-(4-nitro-m-tolyl) phosphorothioate] is an organophosphate insecticide that has been shown to have antiandrogenic activity using in vitro and in vivo screening assays. Studies were performed to evaluate the ability of fenitrothion to disrupt androgen-dependent sexual differentiation in the male rat. Pregnant Crl:CD(SD)BR rats were administered fenitrothion by gavage at 0, 5, 10, 15, 20, or 25 mg/kg/day ( n = 6–11/group) from gestation day (GD) 12 to 21. Maternal toxicity was observed in the dams treated with 20 and 25 mg fenitrothion/kg/day based on muscle tremors and decreases in body weight gain from GD 12 to 21. Fetal death was increased in the 20 and 25 mg/kg/day exposure groups, as evidenced by a decrease in the proportion of pups born alive. Androgen-mediated development of the reproductive tract was altered in male offspring exposed in utero to maternally toxic levels of fenitrothion (25 mg/kg/day), as evidenced by reduction in anogenital distance on postnatal day (PND) 1 and retention of areolae on PND 13. However, these effects were only transient, and there were no indications of abnormal phenotypes or development of androgen-dependent tissues on PND 100. At the dose levels evaluated in this study, fenitrothion was only weakly antiandrogenic in vivo compared with other androgen receptor antagonists such as flutamide, linuron, and vinclozolin. Based on observed fetotoxicity at 20 mg/kg/day, the lowest observed adverse effect level (LOAEL) for developmental effects can be lowered from 25 to 20 mg/kg/day.

Key Words: fenitrothion; reproductive development; androgen receptor antagonist; in utero exposure; anogenital distance; nipple retention.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of chemicals in the environment that have antiandrogenic activity and thus the ability to disrupt the endocrine system is a source of concern (Kelce et al., 1998Go), as androgens are critical for male sexual differentiation (George and Wilson, 1994Go). Testosterone is required for differentiation of the Wolffian duct into the epididymis, vas deferens, and seminal vesicles, as well as for testicular descent. Conversion of testosterone to the more potent androgen dihydrotestosterone (DHT) by 5{alpha}-reductase is required for differentiation of the prostate and external genitalia. Antiandrogens can disrupt male sexual differentiation by several mechanisms, including antagonism of receptor binding, or by inhibition of the production, transport, or metabolism of androgens (LeBlanc et al., 1997Go). Among the pesticides known to competitively bind to the androgen receptor are metabolites of the fungicide vinclozolin (Kelce et al., 1994Go; 1997Go), the DDT metabolite, p,p`-DDE (Kelce et al., 1995Go), the herbicide linuron (Lambright et al., 2000Go; McIntyre et al., 2000Go), and the fungicide procymidone (Ostby et al., 1999Go). More recently, some of the phthalate esters, including di(n-butyl) phthalate (DBP) and diethylhexyl phthalate (DEHP), have been shown to disrupt androgen-mediated development in rats (Gray et al., 2000Go; Mylchreest et al., 1999Go, Gray et al., 1999aGo,bGo; McIntyre et al., 2000Go, 2001aGo; Mylchreest et al., 1998Go, 1999Go, 2000Go; You et al., 1998Go). The incidence and severity of the effects observed depends on the mechanism of action of the chemical, dose level, and timing of exposure. Effects in the male offspring can include a reduction of anogenital distance (AGD), retained areolae/nipples, hypospadias, undescended testes, lesioned testes, malformed epididymides, and small to absent sex accessory glands. In addition, organ weights of androgen-dependent tissues such as the testis, epididymis, prostate, seminal vesicles, and levator ani bulbocavernosus (LABC) muscle are also decreased.

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, 2001Go). 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, 1998Go; U.S. EPA, 1998Go). 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., 1999Go; Landrigan et al., 1999Go; Lu et al., 2001Go). Organophosphate insecticides are well-characterized neurotoxins; they inhibit cholinesterase activity and induce cholinergic stress (Pope, 1999Go). 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., 2001Go). 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., 2001Go). This showed that fenitrothion is comparable to flutamide in potency (KB value of 1.07 x 10-8M) (Maness et al., 1998Go) and 30-fold more potent than linuron (KB value of 75.8 x 10-8M) (McIntyre et al., 2000Go). 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., 2001Go).

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., 2001Go), 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
This study was conducted in accordance with federal guidelines for the care and use of laboratory animals (National Research Council, 1996Go) and was approved by the Institutional Animal Care and Use Committee of the CIIT Centers for Health Research (CIIT). Time-mated, 8- to 10-week-old, nulliparous Crl:CD(SD)BR rats were obtained from Charles River Laboratories Inc. (Raleigh, NC) on GD 0. GD 0 was defined as the day sperm was found in the vagina of the mated female. Animal allocation to treatment groups was done by body weight randomization. Animals were housed in the CIIT animal care facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International, in a HEPA-filtered, mass air-displacement room with a 12-h light-dark cycle at 18–26°C and relative humidity of 30–70%. Animals had access to deionized water and rodent chow ad libitum (NIH-07, Zeigler Brothers, Gardner, PA). Individual dams and offspring were housed in polycarbonate cages on ALPHA-dri bedding (Shepherd Specialty Papers, Kalamazoo, MI) until weaning (postnatal day [PND] 21), at which time animals were ear-tagged and housed by sex and treatment in groups of up to four per cage. At PND 45, all male littermates were separated into two or three per cage until necropsy.

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., 2000Go; Moser et al., 1988Go). 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 (60–65 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 (96–105 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Fenitrothion on the Dams
Dams treated with fenitrothion showed signs of toxicity in the two highest dose groups (20 and 25 mg/kg/day), as evidenced by a significant decrease (14% and 19%, respectively) in body weight gain between GD 12 and 21 (Table 1Go) and adverse clinical signs. No clinical signs of toxicity were observed in the abbreviated FOBs performed on the first day of dosing. However, the dams dosed with 20 and 25 mg/kg/day fenitrothion on GD 17 showed signs of cholinergic stress within 2 h of being treated. An abbreviated FOB performed on GD 19 confirmed that there was a significant effect on a variety of parameters, including gait, open-field tremors, and acoustic response in animals exposed to 20 and 25 mg/kg/day fenitrothion (p < 0.01; data not shown). Severity of the clinical signs increased with subsequent days of dosing such that dams in both the dosed groups displayed eye bulging, lacrimation, and porphyria around the eyes and nose. However, all dams successfully gave birth to a litter, and the length of gestation was not affected by treatment. Dam food consumption and body and organ weights (kidney, liver, and uterus) were not significantly altered by late gestational exposure to fenitrothion (Table 1Go).


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TABLE 1 Parameters in Pregnant Female Rats Treated with Fenitrothion during GD 12–21
 
Effects of Fenitrothion on Reproductive Performance
Developmental toxicity was observed in the rats exposed in utero to fenitrothion, as evidenced by a significant decrease in the number of live pups per litter in the 25 mg/kg/day group and in the proportion of pups born alive in the 20 and 25 mg/kg/day groups (Table 2Go). The number of implantation sites was similar for all the groups, indicating that the decrease in live pups was a result of postimplantation losses. One control dam gave birth to a litter of three female pups that was excluded from the analysis. One animal in the 15 mg/kg/day group gave birth to a litter in which all the pups died by PND 4, and another animal in the 25 mg/kg/day group gave birth to a litter of dead pups. Survival of pups through to weaning, sex ratio, and individual pup weight were not significantly affected by fenitrothion exposure (Table 2Go).


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TABLE 2 Reproductive Parameters in Rats Exposed to Fenitrothion during GD 12–21
 
Effects of in Utero Fenitrothion on the Male Offspring
Late gestational exposure (GD 12–21) to fenitrothion resulted in a decrease in AGD (adjusted for body weight by covariate analysis) in the F1 males at PND 1. The 16% decrease in the 25 mg/kg/day group was highly significant, while the 8% decrease in AGD in the 20 mg/kg/day dose group approached significance (Fig. 1Go). Fenitrothion exposure did not alter AGD of the female littermates. Exposure to fenitrothion in utero also resulted in a significant increase in the incidence of retained areolae in the F1, males exposed to 25 mg fenitrothion/kg/day compared with the control group (litter means were 1.25 and 4.31 in the control and fenitrothion-exposed groups, respectively; Fig. 2Go). The mean number of areolae per litter was not significantly different from controls in the other fenitrothion treatment groups (0.76, 0.37, 1.4, and 1.9 in the 5, 10, 15, and 20 mg fenitrothion/kg/day groups, respectively). In addition, contingency analysis of areolae retention based on the presence or absence of areolae gave similar results such that the male pups in the highest dose group showed a statistically significance increase in response compared with the control group (p < 0.001; data not shown). Litter means for age at preputial separation were unaltered by fenitrothion exposure and were 44.1 0.3, 43.6 ± 0.4, 43.4 ± 0.4, 43.2 ± 0.4, 44.6 ± 0.4, and 44.1 ± 0.5 for the 0, 5, 10, 15, 20, and 25 mg/kg/day fenitrothion-exposed groups, respectively. One animal in the 5 mg/kg/day fenitrothion-exposed group displayed undescended testes, whereas no other malformations of the external genitalia were observed in the any of the other male offspring. This effect on testicular descent in one animal in the lowest dose group is unlikely to be related to late gestational exposure to fenitrothion.



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FIG. 1. Effect of prenatal fenitrothion exposure from gestation day 12 to 21 on AGD on PND 1 and PND 100. Results are presented as nested litter means ± SE. Body weight was used as a covariate. For comparison, the litter mean for female AGD on PND 1was 0.86 ± 0.039 in the control group and this was not altered by fenitrothion exposure. ***Significantly different from control, p < 0.001; n = 10 control litters and 5–7 fenitrothion-exposed litters.

 


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FIG. 2. Effect of prenatal fenitrothion exposure from GD 12 to 21 on areola retention in male rats on PND 13. Results are presented as nested litter means ± SE. Note that none of the male rats displayed nipples on PND 100. ***Significantly different from control, p < 0.001; n = 10 control litters and 5–7 fenitrothion-exposed litters.

 
Male offspring necropsied on PND 96–105 did not show a dose-responsive increase in the incidence of macroscopic testicular and/or epididymal lesions. One animal in the control group displayed a unilateral hypoplastic testis associated with a hypoplastic epididymis. The one animal in the 5 mg/kg/day fenitrothion-exposed group with bilateral cryptorchidism had atrophic testes and hypoplastic epididymides. One rat in the 15 mg/kg/day group and two animals in the 20 mg/kg/day group were observed to have smaller testes than normal associated with hypoplastic epididymides. In addition, two animals in the control group, and one each in the 15 and 20 mg/kg/day fenitrothion-exposed groups had unilaterally enlarged testes compared with the control rats, as indicated by organ weight and gross observation. The grossly atrophic testes from all groups contained tubules with seminiferous epithelial degeneration. The lesion varied from approximately 25% of the tubules affected to all tubules in several of the testes having some degree of alteration. The degenerative epithelium had decreased to completely absent germ cells with small numbers of multinucleated germ cells and cellular debris. Additionally, the tubular lumina were smaller in these atrophic testes. The testes from the cryptorchid animal had similar findings consistent with chronic cryptorchidism. The epididymides contained decreased amounts to no sperm and variable amounts of cellular debris. The lumen of the epididymal duct was often shrunken and the epithelium of the distal body and tail thickened with cribiform change. The seminiferous tubules in the enlarged testes were consistently dilated with increased luminal diameter and variable atrophy of the seminiferous epithelium. The epididymides associated with these testes were normal or contained slightly increased amounts of cell debris.

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. 1Go), 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 3Go). 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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TABLE 3 Organ Weights of PND 100 Male Rats Exposed in Utero to Fenitrothion during GD– 21
 
Effects of in Utero Fenitrothion Exposure on the Female Offspring
In utero exposure to fenitrothion had no effect on the age of onset of vaginal opening; litter means ± SE were 31.1 ± 0.3, 31.2 ± 0.4, 30.9 ± 0.3, 30.5 ± 0.4, 31.0 ± 0.4, and 31.6 ± 0.4 for the 0, 5, 10, 15, 20, and 25 mg/kg/day fenitrothion-exposed groups, respectively. Female offspring necropsied on PND 60–65 did not show a dose-dependent alteration in either body weights or organ weights (data not shown). Liver weight in the 5 mg/kg/day group was significantly decreased by 5% relative to the control group. Nested litter means for the female offspring liver weights were 10.63 ± 0.14, 10.05 ± 0.18, 10.55 ± 0.17, 10.54 ± 0.18, 11.09 ± 0.19, and 10.96 ± 0.20 g for the 0, 5, 10, 15, 20, and 25 mg/kg/day fenitrothion groups, respectively. Kidney weight in the 15 mg/kg/day group was significantly decreased by 8% compared with the control group; nested litter weight means were 2.09 ± 0.04, 2.02 ± 0.05, 1.97 ± 0.05, 1.92 ± 0.05, 2.16 ± 0.05, and 2.06 ± 0.06 g for the 0, 5, 10, 15, 20, and 25 mg/kg/day groups, respectively. However, as the data for the other groups were similar to the controls, these reductions in organ weights are unlikely to be treatment-related.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main impetus behind this study was the finding by Tamura et al. (2001) that fenitrothion antagonized the effects of testosterone propionate (TP) on androgen-dependent organ weights in a Hershberger assay, thus demonstrating its activity as an antiandrogen in vivo. Several laboratories have performed Hershberger assays to evaluate whether fenitrothion has antiandrogenic activity in vivo, with differing results (Sohoni et al., 2001Go; Sunami et al., 2000Go; Tamura et al., 2001Go). EDSTAC has recommended a standard protocol for the Hershberger assay (U.S. EPA, 1998Go), as its sensitivity is influenced by the age of the animal at castration (Lambright et al., 2000Go), relative dose levels of fenitrothion and TP, and length of dosing. In line with EDSTAC recommendations, Tamura et al. (2001) used immature castrated rats and tested the ability of 15 and 30 mg/kg/day fenitrothion administered daily for 7 days to inhibit the effects of 50 µg/day TP on seminal vesicle, LABC, and ventral prostate weights. Both doses of fenitrothion induced significant decreases in the absolute weights of these androgen-responsive tissues; the decreases were also significant when the analysis was performed using terminal body weight as a covariate. Sohoni et al. (2001) used adult castrate rats and administered either 10 or 15 mg/kg/day fenitrothion for 10 days in combination with 0.4 mg/kg/day TP. Neither dose level of fenitrothion was able to compete against the effects on organ weights induced by very much higher doses of TP. Similarly, Sunami et al. (2000) were unable to demonstrate any antiandrogenic activity of fenitrothion when adult castrated rats were administered 0.75, 1.5, or 3 mg/kg/day fenitrothion for 5 days in combination with even higher doses of TP (1 mg/kg/day) than those used by either Tamura et al. (2001) or Sohoni et al. (2001). The contradictory information from the Hershberger assays resulting from procedural differences among studies demonstrated the need to perform a more detailed evaluation of the dose-response characteristics of fenitrothion in vivo.

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., 1990Go; Gray et al., 1999aGo,bGo; Hellwig et al., 2000Go; McIntyre et al., 2000Go, 2001aGo, bGo; Mylchreest et al., 1999Go, 2000Go; Ostby et al., 1999Go; Parks et al., 2000Go). 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., 2000Go, 2001bGo) and to 250 mg/kg/day DBP (Mylchreest et al., 1999Go, 2000Go). 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 3–4 areolae/pup compared with less than one areola in the control group (McIntyre et al., 2000Go, 2001bGo).

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., 2001bGo). Transient effects have been described in male offspring exposed to low dose levels of finasteride (Clark et al., 1990Go) and vinclozolin (Hellwig et al., 2000Go). 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., 2000Go). 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, 1997Go; U.S. EPA, 1997Go). 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, 1998Go). 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., 2001Go).

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, 1997Go). 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., 1989Go). 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, 1997Go). 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, 1997Go). 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, 1997Go). 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, 1994Go). 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, 1994Go). 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., 1988Go; Sultatos, 1994Go), 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, 1989Go), or to a metabolite that maintains its antiandrogenic activity, albeit weaker than the parent compound as for linuron (Cook et al., 1993Go). 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.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Barry McIntyre for his helpful advice, Drs. Susan Borghoff and Jeffrey Everitt for critical review of this manuscript, Dr. Barbara Kuyper for editorial review, and Ms. Sadie Smith-Leak for assistance in manuscript preparation. The authors are very grateful to Mr. Paul Ross and the animal care staff, and to Ms. Elizabeth Gross Bermudez and the necropsy and histology staff. This study was made possible by funds from the American Chemistry Council.


    NOTES
 
1 To whom all correspondence should be addressed. Fax: (919) 558-1300. E-mail: kturner{at}ciit.org. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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