In utero exposure to simvastatin reduces postnatal survival and permanently alters reproductive tract development in the Crl:CD(SD) male rat

Toxicology and Applied Pharmacology 365 (2019) 112–123

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In utero exposure to simvastatin reduces postnatal survival and permanently alters reproductive tract development in the Crl:CD(SD) male rat

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Brandiese E.J. Beverlya,b,1, Johnathan R. Furra,1, Christy S. Lambrighta,1, Vickie S. Wilsona,1, ⁎ Barry S. McIntyrec, Paul M.D. Fosterc, Greg Travlosc, L. Earl Gray Jr.a, ,1 a

Reproductive Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, B105-04, 109 TW Alexander Dr., Research Triangle Park, NC 27709, United States b Oak Ridge Institute for Science and Education, Oak Ridge, TN 37831, United States c National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, United States

ARTICLE INFO

ABSTRACT

Keywords: Simvastatin (SMV) Postnatal survival Male rat sex differentiation

We showed previously that in utero exposure to the cholesterol-lowering drug simvastatin (SMV) during sex differentiation lowers fetal lipids and testicular testosterone production (T Prod) in Hsd:SD rats. Here, the effects of SMV on fetal lipids and T Prod in Crl:CD(SD) rats were correlated with postnatal alterations in F1 males. The current study was conducted in two parts: 1) a prenatal assessment to confirm and further characterize the dose response relationship among previously reported alterations of SMV on fetal T Prod and the fetal lipid profile and 2) a postnatal assessment to determine the effects of SMV exposure during the periods of major organogenesis and/or sexual differentiation on F1 offspring growth and development. We hypothesized that SMV would have adverse effects on postnatal development and sexual differentiation as a consequence of the disruptions of fetal lipid levels and testicular T Prod since fetal cholesterol is essential for normal intrauterine growth and development and steroid synthesis. In the prenatal assessment, SMV was administered orally at 0, 15.6, 31.25, 62.5, 80, 90, 100, and 110 mg SMV/kg/d from GD 14-18, the period that cover the critical window of sex differentiation in the male rat fetus. T Prod was maximally reduced by ~40% at 62.5 mg/kg/d, and higher doses induced overt maternal and toxicity. In the postnatal assessment, SMV was administered at 0, 15.6, 31.25, and 62.5 mg/kg/d from GD 8–18 to determine if it altered postnatal development. We found that exposure during this time frame to 62.5 mg SMV/ kg/d reduced pup viability by 92%, decreased neonatal anogenital distance, and altered testis histology and morphology in 17% of the F1 males. In another group, SMV was administered only during the masculinizing window (GD14-18) at 62.5 mg/kg/d to determine if male rat sexual differentiation and postnatal reproductive development were altered. SMV-exposed F1 males displayed female-like areolae/nipples, delayed puberty, and reduced seminal vesicle and levator ani-bulbocavernosus weights. Together, these results demonstrate that in utero exposure to SMV reduces offspring viability and permanently disrupts reproductive tract development in the male offspring. While the effects of high dose, short term in utero exposure to SMV in the adult male are likely androgen-dependent and consistent with the 40% reduction in T Prod in the fetal testes, long-term, lower dose administration induced some effects that were likely not mediated by decreased T Prod.

1. Introduction SMV is a member of the statin family of cholesterol-lowering drugs

that inhibits 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step of the cholesterol biosynthetic pathway. As cholesterol is a precursor for steroid biosynthesis, inhibition of the

Corresponding author at: Mail Code B105-04, 109 TW Alexander Dr., Research Triangle Park, NC 27709, United States. E-mail addresses: [email protected] (B.E.J. Beverly), [email protected] (J.R. Furr), [email protected] (C.S. Lambright), [email protected] (V.S. Wilson), [email protected] (B.S. McIntyre), [email protected] (P.M.D. Foster), [email protected] (G. Travlos), [email protected] (L. Earl Gray). 1 Present address: National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27,709. ⁎

https://doi.org/10.1016/j.taap.2019.01.001 Received 27 October 2018; Accepted 1 January 2019 Available online 11 January 2019 0041-008X/ © 2019 Elsevier Inc. All rights reserved.

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cholesterol biosynthetic pathway in utero would be expected to disrupt embryo/fetal development and reduce testosterone production (T Prod) in the fetal testes. Disruption of the cholesterol synthesis pathway has profound effects on mammalian embryo and fetal development. Although studies have reported conflicting data on the teratogenicity of statins in rodents and rabbits (Dostal et al., 1994; Henck et al., 1998; Minsker et al., 1983) (FDA, 2005), it was recently reported that administration of SMV at 20 mg/kg/d throughout pregnancy induced significant fetal wastage in the rat (Aditya, 2014). Previously, we demonstrated that SMV reduces T Prod through inhibition of cholesterol synthesis without altering steroidogenic, Insl3, or steroid transport gene expression levels in the Harlan SD rat (Beverly et al., 2014). Androgen signaling during sex differentiation in mammals is a critical pathway in the morphological differentiation of the male reproductive tract. T Prod by fetal Leydig cells is necessary for differentiation of the Wolffian structures (e.g. epididymis, vas deferens, and seminal vesicles), whereas masculinization of the external genitalia, nipple anlagen and prostate is mediated by dihydrotestosterone, a metabolite of testosterone. Since major reductions in testicular T Prod during the masculinization window in the fetal rat can cause reproductive tract malformations (Barlow and Foster, 2003; Gray Jr. et al., 2016; Howdeshell et al., 2015; Mylchreest et al., 1998; Mylchreest et al., 2000; Parks et al., 2000), we hypothesized that SMV, which reduces T Prod by < 50%, would result in subtle alterations of F1 male rat sexual differentiation and reproductive tract tissues that would induce few if any malformations of the reproductive tract. The objectives evaluated in the current study were 1) to confirm and further characterize the potential dose response relationship among previously reported alterations of SMV on fetal T Prod and the fetal lipid profile in a different rat strain (Charles River Sprague Dawley Crl:CD(SD) rats), 2) to determine if administration of SMV during GD 14 to 18, the period of sexual differentiation, produced permanent reproductive tract alterations in the male offspring and 3) to determine if administration of SMV throughout pregnancy, particularly during the periods of major organogenesis and sexual differentiation, would induce adverse effects on F1 offspring growth and development.

purity = 99%) was provided by the National Toxicology Program (Research Triangle Park, NC), and corn oil (CAS 8001-30B7) was purchased from Sigma-Aldrich Corp (St. Louis, MO). 2.3. Prenatal experiment 2.3.1. Doses and administration of chemicals Prenatal assessments were conducted in several blocks, with approximately 12–15 dams per block unless otherwise noted. Dams in each block were ranked by body weight and assigned to groups, with 3–4 dams per group, and each group having similar weights and variances. The dose ranges for SMV were selected based on a previous study conducted in our laboratory using the Harlan (Hsd:SD) SD rat (Beverly et al., 2014). Time-mated rats were dosed by oral gavage with vehicle (water, 2.5 ml/kg maternal body weight) control, 15.6, 31.25, 60, 80, 90, 100, or 110 mg SMV/kg/d from GD 14-18, the period that covers the critical window of sex differentiation in the male rat fetus (Carruthers and Foster, 2005; Wolf et al., 2000). As a point of reference, online Food and Drug Administration (FDA) documents indicate that a dose of 25 mg/ kg/d in male rats results in a mean plasma drug level approximately 4 times higher than the maximum human exposure level in patients given an 80 mg oral dose based on mg/m2 surface area (http://www.drugs. com/pro/simvastatin.html). 2.3.2. Maternal and fetal necropsies Several hours after dosing on GD 18, dams were euthanized by decapitation. Maternal trunk blood was collected into serum separator vacutainer tubes, centrifuged and serum stored at 4 °C for subsequent progesterone analyses and liver weights were taken. All necropsies were conducted within a two hour time frame between 08:00 and 10:00 am Eastern Standard Time to avoid any potential confounding effects of fetal growth or time of day on the fetal endpoints. In two of the blocks, maternal serum also was used for lipid profiling and fetal trunk blood was collected in heparinized capillary tubes and plasma pooled by gender for analysis of the lipid profile, as described previously (Beverly et al., 2014).

2. Materials and methods

2.3.3. Ex vivo fetal testicular T Prod Fetal testes were removed (one testis from three males per litter, n = 3 per litter) and immediately transferred to a single well on a 24well plate (one testis per well) containing 500 μl M-199 media without phenol red (Hazelton Biologics, Inc., St. Lenexa, KS), supplemented with 10% dextran-coated charcoal-stripped fetal bovine serum (GE Healthcare Life Sciences, HyClone Laboratories, Logan, UT). The experiment was conducted using 22, 8, 8, 22, 3, 1, 3, 2, 3 litters in the 0, 15.6, 32.25, 62.5, 80, 90, 100 and 110 mg SMV/kg/d dose groups. Dosage levels at 80 mg/kg/d and above were only included in a single block since these dose levels reduced maternal weight gain during dosing. Testes were incubated in a humidified atmosphere for 3 h at 37 °C on a rotating platform. Following incubation, media were removed and stored at −80 °C until analyzed for testosterone (T). The level of T in the media was measured by radioimmunoassay (RIA) according to the manufacturer's instructions (Diagnostic Products Corporation Coat-A-Count kits, Siemens Corp, Los Angeles, CA). The intra-assay coefficient of variation was 3.1% (based on variability of the standard curve), and the inter-assay coefficient of variation was 13.7%. Cross-reactivity of the T antibody with dihydrotestosterone (DHT) was 3.2%. The limit of detection was 0.2 ng/ml for T.

2.1. Animals This study was reviewed and approved by the USEPA National Health and Environmental Effects Research Laboratory's Institutional Animal Care and Use Committee. Time-mated Sprague Dawley Crl:CD (SD) rats of approximately 90 days of age were purchased from Charles River Laboratories. Sperm positive females were shipped to the US EPA on gestational day (GD) 2 (the day after mating is designated as GD 1). Upon arrival at the EPA, dams were housed individually in clear polycarbonate cages (20 × 25 × 47 cm) with laboratory-grade pine shavings as bedding in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The temperature within the facility was maintained at 20–22 °C with 45–55% humidity, and a 12:12 h. photo period (light/dark cycle, lights off at 7:00 pm). Dams and offspring were provided with NIH07 rat chow during gestation and lactation and NTP 2000 after weaning. Rats had access to filtered (5 μm) municipal tap water ad libitum which is tested every 4 months for a subset of heavy metals, pesticides, and other chemical contaminants and tested monthly for Pseudomonas. 2.2. Chemicals

2.3.4. Lipid profiles Evaluations of lipid concentrations in maternal serum and fetal plasma (n = 7–8 dams/litters per dose group) were performed using an Olympus AU400e chemistry analyzer (Beckman Coulter, Inc., Irving,

SMV (CAS 79902-63-9; purity: 98%; Lot # 11B7D41K) was purchased from AK Scientific (Union City, CA), Dipentyl phthalate (DPeP; CAS 131-18-0; lot# 1431420; RTI Log No. 040109-A-14; Chem ID K43;

113

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TX). Reagents for total cholesterol and triglycerides were obtained from the manufacturer, and reagents for high density lipoprotein (HDL) and low density lipoprotein (LDL) analyses were obtained from Diazyme Laboratories (Poway, CA).

HDL, LDL, and triglycerides) and biomarkers of liver (total protein, albumin, direct and total bilirubin, total bile acids, alanine transaminase, aspartate transaminase and alkaline phosphatase) and kidney (creatinine and blood urea nitrogen) function. The ventral surface of the male rats was shaved and examined for abnormalities including nipple retention (number and location of retained nipples) and external malformations (hypospadias, abnormal glans penis, and vaginal pouch). Internal gross reproductive tract findings including epididymal agenesis, gubernacular agenesis or elongation (length measured in mm with Vernier calipers), testicular malformations (fluid-filled testes, undescended testes, etc.), and ventral prostate or seminal vesicle agenesis were also assessed and documented. The weights of the kidney, liver, ventral prostate, levator ani bulbocavernosus (LABC) muscle complex, seminal vesicles/coagulating gland (with fluids) were recorded. Sperm were collected from one cauda epididymis and a caput plus corpus epididymis from x? males/ litter, where possible, to determine epididymal sperm reserves as previously described (Gray Jr. et al., 1989).

2.3.5. Maternal serum progesterone analysis Progesterone concentrations in maternal serum (n = 7–8 dams/litters per dose group) were measured by RIA according to manufacturer's instructions (Coat-A-Count Kits, Seimens Corp., Los Angeles, CA). The limit of detection was 0.02 ng/ml for progesterone. 2.4. Postnatal experiments Postnatal experiments included two modified one-generation protocol studies with in utero exposure. In the first, a dose response study, SMV was administered at several dosage levels from GD 8 to 18 to determine if in utero statin exposure during the period of major organogenesis and sexual differentiation altered postnatal development in male rats. In the second study, SMV was administered only during the masculinizing window (GD14 to 18) to determine the biological relevance of the observed reduction in GD 18 T Prod, and if this reduction was associated with any permanent male reproductive tract abnormalities that become evident later in life. The second study utilized a single selected SMV dose level since lower exposure levels had minimal effects on T Prod or reproductive development and dose levels above 62.5 mg/kg/d did not further reduce T Prod but induced appreciable fetal and maternal toxicity.

2.4.3. Histopathology of testes and epididymides from F1 males Paired testes and one epididymis from 3, 6, and 3 males from the control, 31.25 SMV/kg (GD8-18), and 62.5 SMV/kg (GD14-18) groups, respectively, were excised, trimmed, fixed in Bouin's solution for 72 h, transferred to 70% ethanol, and submitted to the National Toxicology Program for histopathological evaluation by a board certified pathologist. In addition, testes and epididymides collected from 17, 18, 13, 4 and 16 other F1 males in the control, 15.6 (GD 8-18), 31.25 (GD 8-18), 62.5 (GD 8-18), and 62.5 (GD 14-18) groups, respectively, were submitted to Experimental Pathology Laboratories, Inc. for histopathological evaluation. Two paraffin blocks per male were sectioned (one block containing a transverse section of both the right and left testes and the other containing a longitudinal section of the epididymis), stained with hematoxylin-eosin (H&E), and examined using light microscopy. In total, testes from 20, 18, 19, 4 and 19 F1 males in the control, 15.6 (GD 8-18), 31.25 (GD 8-18), 62.5 (GD 8-18), and 62.5 (GD 14-18) groups, respectively were examined for histopathological lesions by board certified pathologists.

2.4.1. Dose levels and administration of chemicals Four groups of time-mated dams (5 per dose group) were dosed daily by oral gavage from GD 8-18, a time period which covers embryofetal organogenesis and sex differentiation in the rat, with 0 (vehicle, water at 2.5 ml/kg maternal body weight), 15.6, 31.25, or 62.5 mg SMV/kg/d. Concurrently, an additional 5 dams were dosed by oral gavage with 62.5 mg SMV/kg/d from GD 14-18 (covering the period of sex differentiation in the rat) to correlate the effects of reduced fetal testicular T Prod with postnatal reproductive tract malformations in the F1 adult male. 2.4.2. Maternal and F1 offspring measurements and necropsies Dams were euthanized after F1 offspring were weaned, and the uteri were removed and the numbers of implantation scars were counted to determine pup survival (percent survival = ((100*number of live pups)/number of implantation scars). Anogenital distance (AGD) in the pups, measured on all pups under a dissecting scope with reticle, and body weight were recorded on postnatal day (PND) 2 (PND 0 was defined as the day the litter was complete). Pups were examined for the presence of areolas (dark focal areas lacking hair) with and without nipple buds on PND 13. Dams and offspring were housed together until weaning on PND 28 at which time pups were separated and housed two per cage by gender with a littermate, where possible. All males were examined for the onset of puberty determined by preputial separation (PPS) beginning at PND 37 and monitored daily until completion. Beginning at PND 120, final body weights were recorded and necropsies focused on male the male reproductive tract were conducted to assess if SMV exposure altered normal development/maturation of the androgen-dependent reproductive tract. F1 offspring were euthanized by decapitation in a separate room within 15 s after removal from the home cage to minimize stress-induced hormone level changes. The order of the necropsy was balanced with respect to the exposure groups. The number of males necropsied in each group were SMV 0: n = 32 males; SMV 15.6: n = 22 males; SMV 31.25: n = 28 males; SMV 62.5: n = 4 males; SMV 62.5 (GD 14-18): n = 23 males. Trunk blood was collected for serum T and clinical chemistry measurements. Clinical chemistry included a lipid profile (cholesterol,

2.4.4. Data analysis and statistics Data analyses for maternal and F1 offspring body weight, progesterone, fetal testicular T Prod, and lipid concentrations were performed as described previously (Beverly et al., 2014). The fetal plasma lipid profile measures were analyzed by pooling the male and female data using a one-way ANOVA. Any fetal or offspring data measured on more than one male per litter were analyzed using litter mean values rather than individual values. Post hoc pairwise comparisons were conducted using the LSMEANS option with PROC GLM when the overall ANOVA model was statistically significant. For the postnatal assessment, data were analyzed using one-way ANOVA through the general linear model procedure in the Statistical Analysis System 9.1 (SAS, SAS Institute, Cary, NC). Post hoc two-tailed ttests were conducted when the overall ANOVA model was significant (p < .05) using LSMEANS procedure. Weight, AGD, and PPS were analyzed using litter means from examination of multiple pups per litter. For these analyses, body weight at two days of age and at weaning were used as covariates for AGD and PPS analyses, respectively. Data on the incidence of malformations in male offspring were analyzed using Fisher's exact probability test. 3. Results 3.1. Prenatal assessment 3.1.1. General toxicity and maternal assessment on GD 18 SMV did not significantly reduce maternal body weight on GD 18 or 114

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Maternal Serum Lipid Profile A

B

Low Density Lipoprotein

High Density Lipoprotein 80

20

Serum Concentration (mg/dl)

*

15

10

5

60

40

20

.5 62

15

31

0

.5 62

5 .2 31

.6 15

0

.2 5

0

0

.6

Serum Concentration (mg/dl)

25

Simvastatin (mg/kg/day)

Simvastatin (mg/kg/day) C

D

Triglycerides

Total Cholesterol 100

Serum Concentration (mg/dl)

300

**

200

100

0

80

*

60

40

20

Simvastatin (mg/kg/day)

5 62 .

31 .2 5

0

.5 62

.2 5 31

.6 15

0

0

15 .6

Serum Concentration (mg/dl)

400

Simvastatin (mg/kg/day)

Fig. 1. Maternal serum lipid panel. Low density lipoprotein (A), high density lipoprotein (B), triglycerides (C), and total cholesterol (D) serum concentrations in Crl:CD(SD) dams receiving 0 (control), 15.6, 31.25, or 62.5 mg SMV/kg body weight/day via oral gavage on GD 14-18. Data represent means ± SE (n = 7 litters for control group; n = 8 litters for treatment groups). *p < .05 versus control; **p < .01 versus control.

alter liver weight at any of the dose levels assessed (data not shown). However, maternal weight gain during dosing was significantly reduced at dose levels ≥ 62.5 mg/kg/d (data not shown). In addition, a few pregnant females in the higher dose groups (80 and 100 mg/kg/d) had no viable fetuses. Maternal serum LDL (p < .05), triglycerides (p < .01), and total cholesterol (p < .05) were significantly reduced on GD18 in the 62.5 mg/kg/d dose, whereas HDL and progesterone concentrations were not affected by SMV administration (Fig. 1). The lipid profile was not measured in females in the higher dose groups (70, 80, 90, 100, and 110 mg SMV/kg/d).

3.1.3. GD 18 ex vivo fetal testicular T Prod Dose response studies were performed to determine the relationship between in utero exposure to SMV and ex vivo testicular T Prod in fetal males at GD 18. In utero exposure to SMV reduced testicular T Prod and was significantly lower than control at all dose levels (p < .001; Fig. 3a). A logistic regression model estimated that T prod plateaued at ~60% of control, resulting in a maximum inhibition of T prod of ~40% compared to control (Fig. 3b). The value midway between the 100% and the lowest T prod (maximum inhibition) and hillslope of the dose response curve (top constrained to 100%, bottom unconstrained) were 8.9 mg/kg/d and −1.59, respectively (Fig. 3b). Because T Prod was not reduced further by SMV doses above 62.5 mg/kg/d and this dose did not affect fetal viability or induce any overt maternal toxicity (7% reduction in maternal body weight on GD18), this dose level was selected for the SMV postnatal study with GD 14 to 18 exposure as well as the maximum exposure for the GD 8 to 18 dosing window.

3.1.2. Fetal lipid concentrations on GD 18 Circulating lipid concentrations on GD 18 were measured in fetal plasma. LDL concentrations were reduced in fetuses exposed to 15.6 and 62.5 mg/kg/d (p < .05 and p < .001 vs control, respectively), and HDL concentrations were reduced fetuses exposed to 31.25 and 62.5 mg/kg/d (p < .01 and p < .001 vs controls, respectively). Total cholesterol was reduced in fetuses exposed to 62.5 mg SMV/kg/d administration (p < .05 vs control), whereas triglyceride levels were not affected at any of the doses tested (Fig. 2). Other clinical measures were unaffected (Suppl table).

3.2. Postnatal assessment 3.2.1. General toxicity Exposure to 15.6 and 31.25 mg/kg/d from GD 8-18 was well tolerated. In the 62.5 mg/kg/d dose group, SMV induced a fetal/neonatal 115

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High Density Lipoprotein

(Absolute Values)

(Absolute Values)

5

00

50

0

0.

62 .

25 31 .

Simvastatin (mg/kg/day)

Simvastatin (mg/kg/day) Triglycerides

Total Cholesterol

(Absolute Values)

0

0.

00

.5 0 62

.2 5 31

0.

15 .6 0

0

20

Simvastatin (mg/kg/day)

.5 0

20

40

62

*

40

60

25

60

80

31 .

Plasma Concentration (mg/dl)

80

00

Plasma Concentration (mg/dl)

(Absolute Values)

0

15 .

0.

60

0

10

50

20

***

62 .

40

**

15

25

***

20

31 .

*

15 .6

60

25

15 .6 0

Plasma Concentration (mg/dl)

Low Density Lipoprotein 80

00

Plasma Concentration (mg/dl)

Fetal Plasma Lipid Profile

Simvastatin (mg/kg/day)

Fig. 2. Fetal plasma lipid panel. Low density lipoprotein (A), high density lipoprotein (B), triglycerides (C), and total cholesterol (D) plasma concentrations in Crl:CD (SD) F1 male fetuses pooled by litter after 5-day in utero exposure to 0 (control), 15.6, 31.25, or 62.5 mg SMV/kg-maternal body weight/day on GD 14-18. Data represent means ± SE. For dam: n = 7 for control and n = 8 for treatment groups. *p < .05, **p < .01, and ***p < .001 versus control.

mortality rate of 92.0 ± 8.0% (Table 1). Because only one litter (containing only four F1 males) in the 62.5 mg/kg/d dose group survived, mortality was the only fetal endpoint that could be statistically analyzed for this group (Table 1). Administration of lower doses of SMV on GD 8-18 or 62.5 mg/kg/d from GD 14-18 did not significantly reduce litter size on PND 2. There was a 14% reduction in perinatal viability in the 31.25 mg/kg/d SMV exposure group but this reduction was not statistically significant (p > .18); it is biologically relevant given the magnitude and the response observed at 62.5 mg/kg (Table 1). Administration of 62.5 mg/kg/d GD 8-18 and GD 14-18 reduced maternal body weight gain by 20–25 g (about 8% of body weight).

offspring (all females had 12 nipples) exposure to 62.5 mg/kg/d SMV from GD 14-18 resulted in a significant increase the percentage of males with areolae/nipples (~40%; p < .01) as well as the number of areolae/nipples per male compared to control at PND13 (p < .05; Fig. 5). Although statistically significant, the mean numbers of nipples/areolae in this dose group was low (~1.2 per male) and these were not permanent since no areolae/nipples were found in any males when necropsied later in life (Table 2). Because of the low survival (one surviving litter containing 4 males) in the 62.5 mg/kg/d (GD 8-18) group, areola/nipple retention could not be analyzed. SMV did not have an effect on body weight in F1 males (Table 2) or females (data not shown).

3.2.2. Anogenital distance, areolae/nipple retention, and body weight in F1 offspring SMV administration significantly reduced AGD in F1 males on PND 2 in the GD 8-18 exposure, 31.25 mg/kg/d dose group (p < .05). There was no significant change in AGDin female offspring (Fig. 4). On PND 13, males and females were examined for retained female-like nipples and/or areolae. While SMV exposure had no effect in the female

3.2.3. Puberty in F1 males Puberty, as measured by the onset of preputial separation (PPS), was monitored from PND 37 until complete. There was a delay in PPS at 62.5 mg/kg/d SMV (GD 14-18) compared to controls, with complete separation occurring at 48.2 ± 1.0 d in this group (versus 45.4 ± 0.2 d for controls; p < .05; Fig. 6). 116

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SMV 31.25 mg/kg/d (GD 8-18) group displayed grossly atrophic and fluid-filled testes (Fig. 7A) and small epididymides (7B). One of the 28 F1 males had both testes affected and 3 others had unilateral testicular malformations (14.3% incidence of gross testicular malformations; p = .042 Fishers Exact test one-tailed; Table 2). (See Fig. 8.) In the 62.5 mg/kg/d (GD 14-18) dose group, seminal vesicle and LABC weights were significantly reduced verses control (p < .05 versus control; Table 2). While the overall effect of SMV was to reduce LABC weights, one male in this group had an extremely large LABC (2.29 g) which was almost twice the size of all the other LABC tissues in this group (1.34 g). While several other androgen-dependent tissues weighed less than control tissues, none of these effects were statistically significant in the 62.5 mg/kg/d (GD 14-18) group (Table 2; Supplemental figure).

GD 14 TO 18 SIMVASTATIN TREATMENT REDUCES TESTIS TESTOSTERONE PRODUCTION

T prod ng/testis

10

8

6

4

2

00 0.

00

3.2.5. Testicular and epididymal histopathology in F1 adult males Four males from three litters in the 31.25 mg/kg/d (GD 8-18) group had fluid-filled atrophic testes and small epididymides (one with bilateral atrophy and three with unilateral atrophy). The tissues from three of those four affected males, along with paired testes and an epididymis from three grossly unaffected males from the same group, three males in the control group, and three males in the 62.5 mg/kg/d (GD 14-18) group were submitted to the NTP for evaluation. Four of the six tissues from males in the 31.25 SMV/kg/d (GD 8-18) dose group displayed abnormal testis histology. Three males in this dose group displayed severe unilateral (n = 2) or bilateral (n = 1) tubular atrophy (one male with a grossly atrophic testis was not saved for histological assessment), interstitial edema surrounded the atrophic tubules (n = 2) and the rete of (n = 1) was slightly dilated and the corresponding epididymides were devoid of sperm, the distal corpus region exhibited contraction with cribriform change, but the structures of the epididymides were otherwise normal. In addition, one male with grossly normal testes in the dose group displayed atrophic tubules with occasional atrophic tubular profiles (4 tubules), adjacent to the rete in one testis. The testis and epididymides from the remaining rats in this group (n = 2 of 6), and the other groups, had no microscopic abnormalities. Histopathological evaluation of the testes and epididymides from the remaining males in this study were evaluated by Experimental Pathology Laboratories, Inc. and all were unremarkable (data not shown for 17, 18, 13, 4, 16 males from the 0, 15.6, 31.25, 62.5 mg/kg/d (GD8–18) and 62.5 mg/kg/d (GD14-18) dose groups, respectively). In summary, of the testes examined for histological alterations, the numbers of affected/normal males in each group were 0/20, 0/18, 4/19 (21%, p < .05), 0/4, 0/16 from the 0, 15.6, 31.25, 62.5 mg/kg/ d (GD8-18) and 62.5 mg/kg/d (GD14-18) dose groups, respectively. In addition, the total numbers of males with gross and/or histological testis lesions in each group out of the number necropsied were 0/32, 0/ 22, 5/28 (17.8% abnormal, p < .02), 0/4, and 0/23 from the 0, 15.6, 31.25, 62.5 mg/kg/d (GD 8-18) and 62.5 mg/kg/d (GD 14-18) dose groups, respectively (Table 2).

11

0

0

.0

0. 10

90

0

.0 80

.5 62

.2 5

0

31

.6 15

0.

00

0

SMV DOSE mg/kg/d

GD 14 TO 18 SIMVASTATIN TREATMENT REDUCES TESTIS TESTOSTERONE PRODUCTION

% CONTROL T prod

110 100 90 80 70 60 50 40 30 1

10

100

SMV DOSE mg/kg/d Fig. 3. Fetal testicular ex vivo testosterone production from Crl:CD(SD) males exposed in utero to SMV during the period of sex differentiation (GD 14-18) following 3-h incubation. The data represent testosterone production as ng/ testis per 3 h and as percent of control. Each point represents the litter mean ± SE. T Prod was significantly reduced in a dose-dependent manner at all dose levels (3a). T inhibition plateaued at ~45% of control (3b). The EC50 and hillslope of the dose response curve were 8.9 mg/kg/d and −1.59, respectively.

3.2.6. Adult F1 male serum clinical chemistry In utero exposure to SMV did not significantly affect any of the clinical chemistry measurements in F1 adults including the lipid profile (cholesterol, HDL, LDL, and triglycerides), biomarkers of liver (total protein, albumin, direct and total bilirubin, total bile acids, alanine transaminase, aspartate transaminase and alkaline phosphatase) and kidney (creatinine and blood urea nitrogen) function (Supplemental Table).

3.2.4. Androgen-dependent organ weights, testosterone levels, sperm counts, gross pathology in F1 adult males Organ weights, T levels, cauda and corpus plus caput epididymal sperm counts, and reproductive organ malformations were measured in a subset of males from all litters in the control and each exposure group. The number of males examined in each group were SMV 0: n = 32 males, five litters; SMV 15.6: n = 22 males, five litters; SMV 31.25: n = 28 males five litters; SMV 62.5: n = 4 males, one litter; SMV 62.5 (GD 14-18): n = 23 males, five litters (Table 2). SMV exposure did not significantly alter T levels, gubernacular cord lengths or epididymal sperm counts (data not shown) consistently in any exposure group. In the SMV 15.6, 31.25 and 62.5 mg SMV/kg/d (GD 8-18) exposure groups, group mean body and organ weights, were not significantly affected by SMV treatment. However, four males in the

4. Discussion In this study, the primary objectives were to determine if GD 8 to 18 SMV administration altered male rat postnatal development or disrupted reproductive development in the male offspring after in utero exposure during organogenesis and/or sex differentiation. Pregnant rats 117

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Table 1 Postnatal Assessment Dose Response: Maternal Weight, Fetal Survival, and Reproductive Tract Malformations in F1 Males Following Oral Administration of Increasing Doses of SMV from GD 8-18 or GD 14-18 Crl:CD(SD) rats.

No. of litters Length of exposure (GD) Maternal body weight (g) Maternal weight gain, GD 8-18 (g) Maternal weight gaina, GD 14-18 (g) No. of live pups

SMV 0 (mg/kg/day) 5 GD 8-18

SMV 15.6 (mg/kg/day) 5 GD 8-18

SMV 31.25 (mg/kg/day) 5 GD 8-18

SMV 62.5 (mg/kg/day) 5 GD 8-18

SMV 62.5 (mg/kg/day) 5 GD 14-18

325.5 ± 10.5

304.5 ± 4.9

326.2 ± 7.2

297.1 ± 6.1

327.0 ± 10.2

85.9 ± 4.0

72.3 ± 3.25

76.1 ± 6.3

54.5 ± 4.1***

N/A

41.04 ± 2.48

34.5 ± 1.3

36.1 ± 3.1

21.7 ± 2.1***

25.6 ± 4.2*

12.0 ± 0.5

10.2 ± 0.4

10.4 ± 1.6

0.8 ± 8.0***

12.0 ± 0.8

***

92.5 ± 3.7

Pup survival (%) Number of F1 males examined

92.6 ± 2.3

93.0 ± 3.4

78.8 ± 12.5

8.0 ± 8.0

32

22

28

4

23

F1 male body weight at PND2 F1 male body weight at PND13

7.9 ± 0.3

7.7 ± 0.3

7.2 ± 0.4

8.1 ± 0.0

7.2 ± 0.4

29.1 ± 0.3

28.6 ± 0.3

26.8 ± 0.5

32.5 ± 1.5

27.5 ± 0.3

Note: Data are means ± SE. Body weight = maternal body weight at GD 18. Maternal weight gain, GD 818 = maternal body weight at GD 18 – maternal body weight at GD 8. aMaternal weight gain, GD 1418 = maternal body weight at GD 18 – maternal body weight at GD 14. Pup survival = (number of live pups at PND2/total implants) × 100. *p < .05 vs SMV 0, ***p < .001 vs SMV 0, SMV 15.6, SMV 31.25, shaded values differ significantly from control.

were administered increasing doses of SMV from GD 8-18 or at a single high dosage level from GD 14-18, and the reproductive development of F1 males was assessed from birth through puberty and adulthood. Several statistically significant adverse reproductive effects were seen in F1 male rats after in utero exposure to SMV including extensive perinatal mortality, reduced AGD, retained nipple/areolae in infants, delayed puberty (PPS), atrophic/fluid filled testis with hypospermatocytogenesis, and reduced androgen-dependent organ weights at adulthood. The occurrence of these effects varied with the dose of SMV and the timing of SMV dosing during gestation. Some of these effects in the F1 male can plausibly be related to reduction in fetal T Prod seen on GD 18 whereas others likely related to the effects of SMV on the fetal lipid profile. The most striking finding in the current study was the observation that in utero exposure to 62.5 mg/kg/d SMV during the periods of organogenesis and sex differentiation (from GD 8–18) reduced F1 viability by 92%. In contrast, SMV was not teratogenic in rats or rabbits at doses (10 and 25 mg/kg/d) that resulted in 3 times the human exposure based on mg/m2 surface area. (http://www.drugs.com/pro/ simvastatin.html). In the current study, lower dosage levels of SMV (15.6 and 31.25 mg/kg/d) administered from GD 8-18 or at 62.5 mg/ kg/ d from GD 14-18 did not significantly affect F1 viability. Similar to our results, Aditya (2014) found that oral administration of SMV from GD 2-20 at 5, 10 and 20 mg/kg/d, 20 mg/kg/d induced complete postimplantation loss. Co-administration of mevalonic acid ameliorated the effect of SMV on fetal loss and also attenuated the reduction in estrogen and progesterone levels seen in the SMV group. However, it is unlikely that a reduction in maternal progesterone levels is causally related to

the fetotoxicity of in utero exposure to SMV because co-administration of a progestin with SMV did not ameliorate the SMV-induced embryonal and fetal loss (Aditya, 2014). In the current study, dosing with SMV from GD 14-18 at dosage levels up to 110 mg/kg/d did not induce fetal mortality or alter maternal serum progesterone levels on GD 18 at any dose level (data not shown). In addition to the SMV-induced F1 mortality, SMV-induced effects on the reproductive tract observed in the male offspring in adulthood also varied with the timing of exposure during development and the dose of SMV. Short-term, high dose (62.5 mg/kg/d) administration during the masculinization programming window (GD 14-18) resulted in anti-androgenic effects that included retained nipples, delayed PPS, and decreased androgen dependent organ weights. These postnatal effects were observed at a dose that caused a ~40% decrease in testicular T Prod in similarly exposed GD 18 fetal males. Longer-term SMV exposure (GD 8-18) which includes the embryonic period of major organogenesis as well as the fetal masculinizing window, induced a low, but significant incidence of testicular abnormalities (14%) and histological alterations, as well as the absence of sperm in the epididymis of male offspring following 31.25 mg/kg/d exposure. High dose, short-term exposure (GD 14-18) SMV effects are likely mediated through SMV's indirect effect on T Prod via disruption of the sterol synthesis pathway by inhibition of HMG-CoA reductase. The effects on nipple retention, PPS, and androgen-dependent organ weights by SMV administration are among the effects that can also be observed after exposure to other chemicals that disrupt androgen signaling during sexual differentiation, including vinclozolin (Gray Jr. et al., 1994; Ostby et al., 1999) and pyrifluquinazon (AR antagonists) some 118

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A

"Nonmonotonic" Effect of Simvastatin

4.0

Short Term dosing

3.8

*

3.6

p<0.02

3.4

*

3.0

40

20

0

0

SIMVASTATIN mg/kg/d Gestational Day 14-18 or 8-18

S .5 62

.5 62

5 .2

15

0

62

.5

.5 0 62

5 31

.2

2 .6 15

0.

00

2.8

**

31

3.2

60

.6

AGD (mm)

Percent of F1 males with

Long Term dosing

Nipples/Areolae at 13 days of age

in utero on CRSD male rat AGD at 2 days of age

B 2.0

Long Term dosing

2.0

1.5

1.0

0.5

*

1.5

1.0

0.5

S .5 62

.5 62

.2 31

0

62 .5 0

5 31 .2

15 .6 2

00 0.

5

0.0

0.0

15 .6

AGD (mm)

Short Term dosing

Per Male

Number of Nipples/Areolae

Female offspring AGD - 2 days of age

Fig. 5. Nipple/areolae retention in Crl:CD(SD) F1 males on postnatal day 13 after in utero exposure to vehicle (per male; data represent means ± SE. Note: Only one litter in the 62.5 dose group; 62.5S = 62.5 mg/kg/day SMV exposure from GD 14-18. *p < .05, **p < .01 versus control.

SIMVASTATIN mg/kg/d Gestational Day 14-18 or 8-18 Fig. 4. Anogenital distance in Crl:CD(SD) males (A) and females (B) at postnatal day 2. Mean anogenital distance in F1 offspring after in utero exposure to vehicle (water), 15.6, 31.25, and 62.5 mg/kg/day SMV from GD8-18 or 62.5 mg/kg/day from GD 14-18. Data represent litter means ± SEM. Note: Only one litter in the 62.5 dose group and no surviving females; 62.5S = 62.5 mg/kg/day SMV short-term exposure from GD 14-18. *p < .02 versus control.

differentiation; events that precede the onset of sexual differentiation of the reproductive tract. Although the current study highlights the consequences of reduced cholesterol on fetal T Prod and the development of the male reproductive tract, the necessity of cholesterol for other organ systems, particularly the nervous system, is well documented (Edmond et al., 1991; Jurevics and Morell, 1995; Jurevics et al., 1997; Turley et al., 1996). De novo synthesis of cholesterol by the fetus is critical for its development, and inborn errors in cholesterol synthesis are associated with significant disruptions of embryonic development (Porter and Herman, 2011). While cholesterol can be obtained through different sources (maternal circulation, cell membranes, de novo synthesis) (Scott et al., 2009), the dam is likely the primary source of cholesterol in early to mid-gestation, and fetal synthesis becomes the main source of cholesterol in mid- to late-gestation (Tint et al., 2006). Given that the maternal cholesterol contribution to the fetus during sexual differentiation is minimal, our results suggest that the SMV-induced reduction in fetal serum cholesterol levels and testis T Prod is due to a direct

phthalate esters (testosterone synthesis disruptorsinhibitors) (Gray Jr et al. 2009; Wilson et al., 2004), and linuron (mixed modes of action) (Beverly et al., 2014; McIntyre et al., 2002). The mechanism by which lower dose, longer exposure (31.25 mg/ kg/d; GD 8-18) SMV exposure induces testicular malformations is less clear. These effects were observed when the exposure window included major organogenesis, and were not observed at the higher dose (62.5 mg/kg/d) when the exposure was limited to the period of sexual differentiation. Furthermore, it seems unlikely that reduced T Prod is a key event in the adverse effects of SMV during organogenesis since the embryo does not produce testosterone from GD 8-14 (Habert and Picon, 1984). It is possible that these testicular lesions are related to alterations of germ cell migration into the gonadal anlagen or gonadal

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Table 2 Postnatal Assessment: Organ and Body Weights and urogenital abnormalities in F1 Male Offspring at Necropsy after In Utero Administration of SMV from GD 8-18 or GD14-18 Crl:CD(SD) rats.

SMV Dose (mg/kg/day) Exposure No. of Litters Body Weight (g)

0

15.6

31.25

62.5

62.5

GD 8-18 5 855.3 ± 27.4 (29) 136.4 ± 5.9 (10) 580.3 ± 56.3 (20) 2.28 ± 0.06 (20) 7.96 ± 0.74 (10) 2.00 ± 0.05 (20) 2.00 ± 0.05 (20) 714.3 ± 13.8 (20) 1.57 ± 0.07 (20) 204.1 ± 24.5 (10) 23.4 ± 1.4 (10) 1.16 ± 0.15 (20)

GD 8-18 5 801.4 ± 14.4 (21) 138.7 ± 5.3 (10) 599.2 ± 54.4 (19) 2.15 ± 0.05 (19) 8.92 ± 0.40 (10) 2.07 ± 0.03 (19) 2.10 ± 0.05 (19) 738.6 ± 15.8 (19) 1.55 ± 0.04 (19) 236.7 ± 16.6 (10) 21.8 ± 0.4 (10)

GD 8-18 1 859.5 (4) 141.0 (4)

1.38 ± 0.23 (18)

GD 8-18 5 827.8 ± 23.5 (28) 129.8 ± 5.3 (10) 553.0 ± 88.3 (19) 2.17 ± 0.09 (19) 9.01 ± 0.57 (10) 1.89 ± 0.06 (19) 1.84 ± 0.09 (19) 669.6 ± 19.5 (19) 1.49 ± 0.03 (19) 192.0 ± 7.8 (10) 23.2 ± 0.8 (10) 1.36 ± 0.20 (20)

1.38 (4)

GD 14-18 5 817.1 ± 35.2 (22) 131.2 ± 4.7 (11) 486.0 ± 39.3 (19) 2.06 ± 0.06* (19) 8.25 ± 0.21 (10) 1.88 ± 0.07 (19) 1.88 ± 0.08 (19) 683.5 ± 19.9 (19) 1.38 ± 0.04* (19) 170.2 ± 13.6 (11) 23.2 ± 0.7 (11) 1.26 ± 0.15 (18)

0

15.6

31.25

62.5

62.5

GD 8-18 32

GD 8-18 21

GD 8-18 28

GD 8-18 4

GD 14-18 22

0 0

0 0

0 0

0 0

0 0

3.1 (1)

0

0

0

0

Agenesis or abnormal seminal vesicle Agenesis or abnormal Gubernaculum Female-like Nipples

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Abnormal Testis

0

0

17.8% * (5/28 males)

0

0

Agenesis of Epididymis

0

0

0

0

0

Undescended Testis Hydronephrosis

0

0

0

0

0

0

4.7 (1)

7.1 (2)

0

0

Glans Penis (mg) Ventral Prostate (mg) Seminal Vesicle (mg) Gubernacular Ligaments (mg) Right Testis (g) Left Testis (g) Epididymis (mean) (mg) LABC (g) Cowper’s Gland (mg) Liver (g) Serum testosterone ng/ml

SMV Dose (mg/kg/day) Exposure Number of males Hypospadias Agenesis of vas deferens Small ventral prostate

597.3 (4) 2.09 (4) 9.9 (4) 1.95 (4) 2.07 (4) 703.9 (4) 1.74 (4) 166.15 (4) 22.9 (4)

Note: Data are mean ± SE; values in () are the number of males used to calculate the litter means. *p < .05 versus control, shaded values differ significantly from control. *P < .02 Fishers Exact one tailed test.

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Age at Preputial Separation (Days)

p<0.03

p<0.06

2.5

48

2.0

46

Weight (g)

n=1

44

62.5

1.0

62.5S

0.5

Dose mg/kg/d Cumulative Frequency distribution of

0.0 0

Effects of in utero SMV on the

100

0 15.6

80

32.5

60

62.5short

Simvastatin (mg/kg/day)

B

Epididymides

62.5long

40

1000

20 0

800 52

54

56

58

60

Age at PPS Fig. 6. Preputial separation in Crl:CD(SD) F1 males after in utero exposure to vehicle (water), 15.6, 31.25, and 62.5 mg/kg/day SMV from GD8-18 or 62.5 mg/kg/day from GD 14-18. Males were examined beginning on postnatal day 37 and monitored daily until separation was complete. The data represent the litter means for each group ± SE. *p < .05 versus control.

600

400

200

0 15

0

effect of SMV on fetal rather than maternal cholesterol synthesis from GD14-18. Although the effects of SMV on T Prod and the fetal lipid profile seen in the current study with the Crl:CD(SD) rat are very similar to what we reported previously in the Hsd:SD rat, there are some notable differences between the effects of SMV on the lipid profile and T Prod on GD 18 in the fetal rat. Crl:CD(SD) control fetal males had significantly (p < .0001) lower plasma triglyceride and cholesterol levels and significantly (P < .0001) higher plasma HDL levels whereas LDL levels did not differ between the control Crl:CD(SD) and Hsd:SD fetal plasma samples (Supplemental File). In addition, SMV treatment decreased triglyceride and cholesterol levels at lower ED50s in the Hsd:SD rat than the Crl:CD(SD) rat whereas HDL and LDL levels were affected similarly in the two rat strains. Comparing T Prod among the two strains, fetal GD 18 Crl:CD(SD) rat testes produced lower levels of T than did the testes from Hsd:SD rats at all SMV dose levels, including the control group. Furthermore, T Prod from testes of Crl:CD(SD) rats declined slightly more rapidly as the dose of SMV increased than the Hsd:SD rat, but the difference did not attain statistical significance (Supplemental figure) when the dose response curves were analyzed using logistic regression models.

.5 S

50

62

48

5

46

.2

44

Weight (mg)

42

31

Percent of Males

with Completel PPS

Age a Full PPS in CRSD F1 male rats

62 .5 S

31.25

31 .2 5

15.6

15 .6

0

1.5

.6

Age in Days

50

Testes

A

Simvastatin (mg/kg/day) Fig. 7. Testicular and epididymal weights in adult F1 males after in utero exposure to SMV. (A) Testicular (A) and Epididymal (B) weights in adult Crl:CD (SD) F1 males after in utero exposure to vehicle, 15.6, or 31.25 mg/kg/day SMV from GD 8-18 or 62.5 mg/kg/day SMV from GD 14-18. Each data point represents an individual organ weight. Note: 62.5S = 62.5 mg/kg/day SMV shortterm exposure from GD 14-18.

In summary, the results of this study clearly demonstrate that maternal exposure to SMV induces adverse effects on fetal viability and permanently alters male rat reproductive development. While the effects of high dose, short term in utero exposure to SMV in the adult male are likely androgen-dependent and consistent with the 40% reduction in T Prod in the fetal testes, long-term, lower dose administration induced some effects that were likely not mediated by decreased T Prod.

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Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), however, the statements, opinions or conclusions contained therein do not necessarily represent the statements, opinions or conclusions of NIEHS, NIH or the United States government. Acknowledgements The authors would like to thank Hunter Sampson and Mary Moody for their assistance during the F1 necropsies and Dr. Dianne Creasy for the comprehensive histopathological analyses of selected testicular and epididymal tissues. Conflicts of interest The authors have no conflicts of interest to report. When the work was performed, the authors all were federal government employees or contractors and funds were provided by the agencies. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.taap.2019.01.001. References Aditya, B.S., 2014. A preliminary study on the reproductive toxicity of statins in rats. Int. J. Res. Biosci. 3, 39–47. Barlow, N.J., Foster, P.M., 2003. Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to di(n-butyl) phthalate. Toxicol. Pathol. 31, 397–410. Beverly, B.E., Lambright, C.S., Furr, J.R., Sampson, H., Wilson, V.S., McIntyre, B.S., et al., 2014. Simvastatin and dipentyl phthalate lower ex vivo testicular testosterone production and exhibit additive effects on testicular testosterone and gene expression via distinct mechanistic pathways in the fetal rat. Toxicol. Sci. 141, 524–537. Carruthers, C.M., Foster, P.M., 2005. Critical window of male reproductive tract development in rats following gestational exposure to di-n-butyl phthalate. Birth Defects Res. B Dev. Reprod Toxicol. 74 (3), 277–285. Dostal, L.A., Schardein, J.L., Anderson, J.A., 1994. Developmental toxicity of the hmg-coa reductase inhibitor, atorvastatin, in rats and rabbits. Teratology 50, 387–394. Edmond, J., Korsak, R.A., Morrow, J.W., Torok-Both, G., Catlin, D.H., 1991. Dietary cholesterol and the origin of cholesterol in the brain of developing rats. J. Nutr. 121, 1323–1330. FDA Cfdear, 2005. Pharmacology review: Simvastatin. 21-961. In: FDA NADA records at drugs.com. Gray Jr LE, Rider CV, Howdeshell KL, Hotchkiss AK, Wilson VS, Foster P, et al., Cumulative effects of administration mixtures of “antiandrogens” in rats: a new framework based upon common systems rather than common mechanisms. In: Proceedings of the Biology of Reproduction, 2009, Soc Study Reprod, 193–193. Gray Jr., L.E., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., et al., 1989. A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundam. Appl. Toxicol. 12, 92–108. Gray Jr., L.E., Ostby, J.S., Kelce, W.R., 1994. Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. 129, 46–52. Gray Jr., L.E., Furr, J., Tatum-Gibbs, K.R., Lambright, C., Sampson, H., Hannas, B.R., et al., 2016. Establishing the "biological relevance" of dipentyl phthalate reductions in fetal rat testosterone production and plasma and testis testosterone levels. Toxicol. Sci. 149, 178–191. Habert, R., Picon, R., 1984. Testosterone, dihydrotestosterone and estradiol-17 beta levels in maternal and fetal plasma and in fetal testes in the rat. J. Steroid Biochem. 21, 193–198. Henck, J.W., Craft, W.R., Black, A., Colgin, J., Anderson, J.A., 1998. Pre- and postnatal toxicity of the hmg-coa reductase inhibitor atorvastatin in rats. Toxicol. Sci. 41, 88–99. Howdeshell, K.L., Rider, C.V., Wilson, V.S., Furr, J.R., Lambright, C.R., Gray Jr., L.E., 2015. Dose addition models based on biologically relevant reductions in fetal testosterone accurately predict postnatal reproductive tract alterations by a phthalate mixture in rats. Toxicol. Sci. 148, 488–502. Jurevics, H., Morell, P., 1995. Cholesterol for synthesis of myelin is made locally, not imported into brain. J. Neurochem. 64, 895–901. Jurevics, H.A., Kidwai, F.Z., Morell, P., 1997. Sources of cholesterol during development of the rat fetus and fetal organs. J. Lipid Res. 38, 723–733. McIntyre, B.S., Barlow, N.J., Foster, P.M., 2002. Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent testicular atrophy. Toxicol. Sci. 65, 62–70. Minsker, D.H., MacDonald, J.S., Robertson, R.T., Bokelman, D.L., 1983. Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme a reductase. Teratology 28,

Fig. 8. Histopathology of adult Crl:CD(SD) rat testes after in utero exposure to vehicle or 31.25 mg/kg/day from GD8-18. (A) Normal histology with normal spermatogenesis in all tubules from a control male. (B) Abnormal testis displaying unilateral severe atrophy with interstitial edema in a male treated with 31.25 mg/kg SMV from GD 8-18. (C) Epididymis from an affected male treated with 31.25 mg/kg SMV from GD 8-18 with normal morphology but no sperm.

Funding information Funding for this project conducted at the USEPA was provided by an interagency agreement between the USEPA and the National Toxicology Program at the National Institute of Environmental Health Sciences (IA: RW-75-92285501-1). B.E.J.B. was funded through an Oak Ridge Institute for Science and Education postdoctoral fellowship. Disclaimers The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory at the U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names of commercial products constitute endorsement or recommendation for use. This research was supported in part by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health. This article may be the work product of an employee or group of employees of the National Institute of 122

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B.E.J. Beverly et al. 449–456. Mylchreest, E., Cattley, R.C., Foster, P.M., 1998. Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: an antiandrogenic mechanism? Toxicol. Sci. 43, 47–60. Mylchreest, E., Wallace, D.G., Cattley, R.C., Foster, P.M., 2000. Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to di(nbutyl) phthalate during late gestation. Toxicol. Sci. 55, 143–151. Ostby, J., Kelce, W.R., Lambright, C., Wolf, C.J., Mann, P., Gray Jr., L.E., 1999. The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro. Toxicol. Ind. Health 15, 80–93. Parks, L.G., Ostby, J.S., Lambright, C.R., Abbott, B.D., Klinefelter, G.R., Barlow, N.J., et al., 2000. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicol. Sci. 58, 339–349. Porter, F.D., Herman, G.E., 2011. Malformation syndromes caused by disorders of

cholesterol synthesis. J. Lipid Res. 52, 6–34. Scott, H.M., Mason, J.I., Sharpe, R.M., 2009. Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. Endocr. Rev. 30, 883–925. Tint, G.S., Yu, H., Shang, Q., Xu, G., Patel, S.B., 2006. The use of the dhcr7 knockout mouse to accurately determine the origin of fetal sterols. J. Lipid Res. 47, 1535–1541. Turley, S.D., Burns, D.K., Rosenfeld, C.R., Dietschy, J.M., 1996. Brain does not utilize low density lipoprotein-cholesterol during fetal and neonatal development in the sheep. J. Lipid Res. 37, 1953–1961. Wilson, V.S., Lambright, C., Furr, J., Ostby, J., Wood, C., Held, G., et al., 2004. Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicol. Lett. 146, 207–215. Wolf, C.J., LeBlanc, G.A., Ostby, J.S., Gray Jr., L.E., 2000. Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicol. Sci. 55, 152–161.

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