Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f(1) sex ratio and mixed results for the sex ratio of the f(2) generation. In order to better understand the potential effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats disrupts brain sexual differentiation

The effects of in utero and lactational exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on brain sexual differentiation were investigated. TCDD was orally administered to pregnant Holtzman rats on gestation day (GD) 15, and the activity of brain aromatase, a key enzyme for sexual differentiation, was measured in offspring on postnatal day (PND) 2. Changes in sexual dimorphisms of saccharin preference and the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were examined in adult offspring. In controls, litter means of brain aromatase activity were higher in males than in females. In utero exposure to 200 ng/kg TCDD significantly decreased the sex ratio of aromatase activity (male/female) on PND 2. Offspring were weaned on PND28 and the saccharin test was started on PND84. In controls, saccharin (0.25%) intake (g/kg body weight) was significantly higher in female offspring than in males. In utero exposure to 200 ng/kg TCDD significantly increased saccharin intake in male offspring compared with control males, whereas 800 ng/kg TCDD had no effect. Neither dose of TCDD influenced saccharin intake of female offspring. In controls, SDN-POA volume was significantly greater in males than in females at 14 weeks of age. Exposure to 200 ng/kg TCDD significantly decreased SDN-POA volume in males, whereas 800 ng/kg TCDD had no effect. Neither doses of TCDD influenced the SDN-POA volume in female offspring. These results suggest that in utero and lactational TCDD exposure dose-dependently induces demasculinization in male offspring by inhibiting brain aromatase activity in the hypothalamus-preoptic area during central nervous system development.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters postnatal development of seminal vesicle epithelium.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to alter male reproductive development of laboratory animals through in utero and lactational exposure. As a result of exposure, the accessory glands of the male reproductive tract, including the seminal vesicle, are decreased in size as determined by total weight of the tissue. Analysis of seminal vesicle weights over time suggests that the changes may be transient. Administration of 1.0 microg/kg TCDD during gestation caused a significant decrease in seminal vesicle weights of offspring 8-11 months of age. We examined the effects of TCDD on seminal vesicles from rats exposed in utero and lactationally. Pregnant Long Evans rats were gavaged on gestation day 15 with 1.0 microg/kg TCDD in corn oil. Male pups were euthanized and necropsied on postnatal days (PND) 15, 25, 32, 49, 63, and 120. Seminal vesicles were weighed and then fixed in 10% neutral buffered formalin and processed for microscopic examination. Seminal vesicle weights were not significantly decreased until PND 32. Androgen receptor mRNA expression in PND 25 seminal vesicles was not different from control. In the present study, TCDD exposure decreased seminal vesicle epithelial branching and differentiation. Control epithelial cells had tall columnar morphology with relatively abundant cytoplasm, whereas TCDD-treated cells had rounded nuclei and less cytoplasm. In addition, immunolocalization of proliferating nuclear antigen was confined to undifferentiated basal epithelial cells of controls but was found in both basal and luminal cells of the treated seminal vesicle. Results indicate that the TCDD-induced impaired growth of the rat seminal vesicles is associated with a dramatic decrease in the development of the epithelium.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone tissue in rhesus monkeys

Bone tissue is one of the target tissues for dioxins and dioxin-like compounds. Therefore, the aim of this study was to investigate effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on bone tissue in rhesus monkey, the most human-like experimental model available. Pregnant rhesus monkeys (Macaca mulatta; age 4-10 years) were exposed to TCDD with a total dose of 40.5-42.0 or 405-420ng/kg bodyweight by repeated subcutaneous injections starting at gestational day 20 and followed by injections every 30 days until 90 days after delivery. At a mean age of 7 years the offspring were sacrificed and the femur bone dissected. Results from peripheral Quantitative Computed Tomography (pQCT) analyses of the metaphyseal part of the femur bones in female offspring showed significant increases in trabecular bone mineral content (BMC; +84.6%, p<0.05, F-value (F)=5.9) in the low-dose treatment group compared with the controls. In the same animals, analysis of the mid-diaphyseal part revealed increases in total BMC (+21.3%, p<0.05, F=5.2) and cortical cross-sectional area (CSA; +16.4%, p<0.01, F=7.4) compared with the controls. In males, changes in biomechanical properties indicating more fragile bone were observed. Displacement at failure were significantly increased in the male low-dose group compared to the controls (+38.0%, p<0.05, F=11). The high dose of TCDD did not induce any significant changes in bone morphology. In conclusion, in utero and lactational low-dose, but not high-dose exposure to 2,3,7,8-TCDD induced disruption of bone tissue development in rhesus monkey, a result suggesting that similar effects might occur in humans also.     

Disruption of thyroid hormone homeostasis at weaning of Holtzman rats by lactational but not in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The purpose of this study is to clarify whether lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is entirely responsible for the perturbation in thyroid hormone homeostasis during the neonatal period. Pregnant Holtzman rats were given a single oral dose of 1.0 mug TCDD/kg body weight on gestational day 15. Half of the litters were cross-fostered with the half of the dams treated with vehicle on postnatal day (PND) 1 to make four groups of rats, control (C/C), prenatal TCDD exposure only (T/C), postnatal TCDD exposure only (C/T), and both prenatal and postnatal TCDD exposure (T/T). On PND 21, the C/T and T/T groups, but not the T/C and C/C groups, showed a significant decrease in serum total thyroxin (TT4) and free thyroxin (FT4) concentrations in both sexes and a significant increase in serum thyroid-stimulating hormone (TSH) levels, particularly male pups. These two groups of male and female pups had significantly higher concentrations of TCDD in the liver, with marked induction of cytochrome P450 (CYP) 1A1 mRNA and intense immunostaining of CYP1A1 in the liver. UDP glycosyltransferase 1 family, polypeptide A6 (UGT1A6) and UGT1A7 mRNAs were induced in their livers, with marked immunostaining of UGT1A6. The transfer of TCDD from dams to the pups was confirmed by the detection of TCDD in mother's milk remaining in the stomachs of lactationally exposed pups on PND 1. The present results demonstrate that lactational, but not in utero, exposure to TCDD was responsible for the disruption of thyroid hormone homeostasis.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects tooth development in rhesus monkeys

We thought to validate the current tolerable daily intake (TDI) value for dioxin (4 pg/kg) in Japan. Pregnant rhesus monkeys received an initial dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0, 30, or 300 ng/kg subcutaneously) on day 20 of gestation; the dams received additional injection of 5% of the initial dose every 30 days until day 90 after delivery. The teeth of stillborn, postnatally dead, and surviving offspring (now approximately 4 years old) were evaluated. None of the offspring in the 0 and 30 ng/kg groups (n=17 and 15, respectively) had tooth abnormalities, whereas 10 of 17 in the 300 ng/kg had them. These findings suggest the lowest-observed adverse-effect level (LOAEL) for TCDD in the rhesus monkey is between 30 and 300 ng/kg, and probably is close to that for rodents (86 ng/kg) on which the current TDI was based. It is reasonable to conclude that the current TDI needs no immediate modification.     

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin causes accelerated terminal differentiation in fetal mouse skin.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental toxin, has been shown to cause a human skin pathology called chloracne. The majority of laboratory mouse strains, with the exception of mice bearing a mutation in thehairless gene, fail to display overt signs of chloracne upon exposure to TCDD. As a result, only minimal data exist on the effects of TCDD in adult haired mice and no data exist on the effects of TCDD in developing mouse skin. Here we report that TCDD affects the temporal expression of protein markers of keratinocyte terminal differentiation during murine skin morphogenesis. Immunohistochemical analysis of E16 mice reveals accelerated expression of the intermediate filament-associated protein filaggrin in response to TCDD. At a later developmental time and after birth, expression of filaggrin and loricrin is indistinguishable between treatment and control groups. At E16 expression of keratins 5, 6, and 10 are unaltered in TCDD-exposed individuals and TUNEL analysis shows no apoptotic cells in the basal and spinous layers of either treatment or control groups. At E16, immunohistochemical analysis of AhR-null mouse skin reveals accelerated filaggrin expression in both vehicle and TCDD exposed animals. We therefore hypothesize that AhR acts as a modulator of late stage keratinocyte terminal differentiation.     

In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status.

When administered in overtly toxic doses to postpubescent rats, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a variety of adverse effects on the male reproductive system including a decrease in plasma androgen concentrations. If such an androgenic deficiency were produced prenatally and/or early postnatally it could potentially impair male reproductive function by disrupting the development of sex organs and/or causing incomplete sexual differentiation of the central nervous system. To determine whether TCDD can reduce androgen concentrations perinatally and/or impair androgen-dependent perinatal development, pregnant Holtzman rats were treated with 1.0 micrograms TCDD/kg or vehicle on Day 15 of gestation. Plasma testosterone concentrations in fetal males were significantly reduced by TCDD on Gestation Days 18 through 21. The surge in plasma testosterone concentrations shortly after birth was also significantly reduced, as was anogenital distance, an androgen-dependent parameter. To further investigate the effects of perinatal TCDD exposure on the male reproductive system, rats born to dams given TCDD (0.064, 0.16, 0.40, or 1.0 micrograms/kg, po) or vehicle on Day 15 of gestation were evaluated from birth through sexual maturation. This report describes their growth, physical development, and androgenic status (i.e., androgen concentrations and androgen-dependent structures and functions); effects on spermatogenesis, testicular histology, sexual behavior, and fertility are reported separately. There was little evidence that TCDD caused maternal toxicity. Signs of overt toxicity in offspring were limited to an 8% reduction in live births (highest dose only) and to decreases in body weight gain and feed consumption (two highest doses only) which disappeared by early adulthood. With respect to androgenic status, maternal TCDD doses as low as 0.16 micrograms/kg produced significant dose-related decreases in the anogenital distance of 1- and 4-day-old males, delays in testicular descent, and decreases in seminal vesicle and ventral prostate weights. The reductions in organ weights were observed when rats were at the juvenile, pubertal, postpubertal, and mature stages of sexual development. Plasma testosterone and 5 alpha-dihydrotestosterone concentrations tended to be reduced at these times (though not significantly), while plasma luteinizing hormone concentrations were generally unaffected. Collectively, these results demonstrate that perinatal TCDD exposure alters the androgenic status of male rats from the fetal stage into adulthood, and that TCDD can affect androgenic status without causing overt toxicity. In rats, the male reproductive system appears to be more sensitive to the toxic effects of in utero and lactational TCDD exposure than any other organ or organ system studied thus far.     

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effects on fetal and adult cardiac gene expression and adult cardiac and renal morphology.

The mouse heart is a target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during fetal development, and microarray analysis demonstrates significant changes in expression of cardiac genes involved in extracellular matrix (ECM) remodeling. We tested the hypothesis that developmental TCDD exposure would disrupt cardiac ECM expression and be associated with changes in cardiac morphology in adulthood. In one study, time-pregnant C57BL/6 mice were dosed with corn oil or 1.5, 3.0, or 6.0 microg TCDD/kg on gestation day (GD) 14.5 and sacrificed on GD 17.5, when changes in fetal cardiac mRNA expression were analyzed using quantitative PCR. TCDD induced mRNA expression of genes associated with ECM remodeling (matrix metalloproteinase 9 and 13, preproendothelin-1 [preproET-1]), cardiac hypertrophy (atrial natriuretic peptide, beta-myosin heavy chain, osteopontin), and aryl hydrocarbon receptor (AHR) activation (cytochrome P4501A1, AHR repressor). Further, all TCDD-induced changes required the AHR since gene expression was not altered in AHR knockout fetuses. In a second study, time-pregnant mice were treated with corn oil or 6.0 microg TCDD/kg on GD 14.5, and male offspring were assessed for changes in cardiac gene expression and cardiac and renal morphology at 3 months. All TCDD-induced changes in cardiac gene expression observed fetally, except for preproET-1, remained induced in the hearts of adult male offspring. Adult male offspring of TCDD-exposed dams also displayed cardiac hypertrophy, decreased plasma volume, and mild hydronephrosis. These results demonstrate that in utero and lactational TCDD exposures alter cardiac gene expression and cardiac and renal morphology in adulthood, which may increase the susceptibility to cardiovascular dysfunction.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in the C57BL/6J mouse prostate: lobe-specific effects on branching morphogenesis.

Branching morphogenesis is an essential component of prostate development. This study was conducted to test the hypothesis that in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure differentially inhibits branching morphogenesis and ductal canalization in the ventral, dorsal, lateral, and anterior mouse prostate. Pregnant C57BL/6J mice were given TCDD (5 microg/kg, orally) or vehicle on gestation day (GD) 13 and their pups examined at 1, 7, 14, 21, 35, and 90 days of age. Prostate lobes were microdissected after incubation in 0.5% collagenase and the numbers of ductal tips, main ducts, and ductal tips per main duct were determined by examining photographs of microdissected, whole-mount specimens. Ductal canalization was determined using histological sections of the dorsolateral and anterior prostate lobes. TCDD inhibited branching morphogenesis in all prostate lobes. The ventral prostate (VP) was extremely small throughout development and never developed any ductal structure. TCDD reduced the numbers of ductal tips and main ducts in the dorsal (DP) and lateral prostate (LP), but reductions in ductal tip numbers appeared to result entirely from reductions in the number of main ducts. Dorsolateral prostate (DLP) weights were slightly reduced by TCDD, but there was no effect on ductal canalization in the dorsal, lateral, or anterior lobes. TCDD had no effect on main duct number in the anterior prostate, but weight, ductal tip number, and the number of ductal tips per main duct was substantially reduced. These results demonstrate that the severe inhibition in ventral prostate development caused by in utero and lactational TCDD exposure is accompanied by a complete absence of branching morphogenesis. The impairment in dorsal, lateral, and anterior prostate (AP) development is associated with a lobe-specific inhibition of the various processes involved in duct formation.     

In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3. Effects on spermatogenesis and reproductive capability.

When administered in overtly toxic doses to postweanling male rats, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces adverse effects on the reproductive system including a decrease in spermatogenesis. Because the male reproductive system may be particularly susceptible to toxic insult during the perinatal period, the effects of in utero and lactational TCDD exposure on its development were examined. Male rats born to dams given TCDD (0.064, 0.16, 0.40, or 1.0 micrograms/kg, po) or vehicle on Day 15 of gestation were evaluated at various stages of development; effects on spermatogenesis and male reproductive capability are reported herein. Testis, epididymis, and cauda epididymis weights were decreased in a dose-related fashion at 32, 49, 63, and 120 days of age, that is, when males were at the juvenile, pubertal, postpubertal, and mature stages of sexual development, respectively. When measured on Days 49, 63, and 120, daily sperm production by the testis was reduced at the highest maternal TCDD dose to 57-74% of the control rate. Cauda epididymal sperm reserves in 63- and 120-day-old males were decreased to as low as 25 and 44%, respectively, of control values, although the motility and morphology of these sperm appeared to be unaffected. The magnitude of the effects described above tended to lessen with time; nevertheless, the decreases in epididymis and cauda epididymis weights, daily sperm production, and cauda epididymal sperm number were statistically significant at the lowest maternal dose tested (0.064 micrograms TCDD/kg) on Day 120 and at most earlier times. To determine if in utero and lactational TCDD exposure also affects male reproductive capability, rats were mated at approximately 70 and 120 days of age with control females. Little if any effect on fertility was seen, and the survival and growth of offspring was unaffected. These results are not inconsistent with the pronounced reductions in daily sperm production and cauda epididymal sperm reserves caused by perinatal TCDD exposure since rats produce and ejaculate far more sperm than are required for normal fertility. The TCDD-induced reduction in spermatogenesis cannot be accounted for by concurrent effects on plasma follicle-stimulating hormone or androgen concentrations or by undernutrition. To investigate the nature of the spermatogenic lesion, leptotene spermatocyte to Sertoli cell ratios were determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat follicular steroidogenesis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant and causes adverse effects on female reproduction when administered to rats. Our aims were to study effects of gestational and lactational exposure to TCDD on ovarian steroidogenesis and steroidogenic enzyme expression of offspring on postnatal day (PND) 14 in the rat and sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro. Synthetic estrogen diethylstilbestrol (DES) was used as a treatment control. Serum progesterone (P4) level in offspring increased significantly on PND 14 in the TCDD (1 microg/kg)-exposed group while body weight, FSH and E2 levels were not changed. In ovarian follicles of offspring on PND 14 in the TCDD-exposed groups, protein expression of cytochrome P-450 aromatase, cytochrome P-450 cholesterol side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxy-steroid-dehydrogenase/Delta(5)-Delta(4) isomerase type 1, or P4 receptor was not affected. TCDD decreased E2 and P4 production in ex vivo follicle culture. DES at a dose level of 0.1mg/kg was dystocic while a dose 0.02 mg/kg increased ovarian ex vivo E2 and testosterone production without affecting P450arom activity indicating stimulation of early steps of steroidogenic pathway. Data suggests that TCDD has multiple targets in ovarian steroidogenesis, but the inhibitory action represented as decreased follicular steroid hormone production ex vivo is not apparent at the ovarian protein expression. Furthermore, TCDD had no direct effect on immature rat ovarian steroidogenesis in vitro suggesting that the follicle culture method is not a sensitive method to study the mechanisms of TCDD action.     

Adult 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and effects on male reproductive organs in three differentially TCDD-susceptible rat lines.

The contribution of genetic factors to adult male reproductive system toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was analyzed in three rat lines differentially resistant to TCDD acute lethality: line A, B, and C rats (selectively bred from TCDD-resistant Han/Wistar [Kuopio; H/W] and TCDD-sensitive Long-Evans [Turku/AB; L-E] rats). The resistance is linked to a mutated H/W-type aryl hydrocarbon receptor allele in line A and to an H/W-type unknown "B" allele in line B. Line C rats do not have resistance alleles. Mature male line A, B and C rats were given single oral doses up to 1000, 300, and 30 micrograms/kg TCDD, respectively. The dose-responses of TCDD effects on male reproductive organ weights, sperm numbers, and serum testosterone concentrations were analyzed 17 days after exposure. Serum testosterone concentrations were decreased by the highest doses of TCDD, and there were no major sensitivity differences among the rat lines. Correspondingly, the decrease in relative weight of ventral prostate and seminal vesicles was seen only after a dose of >/=100 micrograms/kg TCDD. Thus the effect was observed only in resistant lines A and B. The relative weights of testes and epididymides were not affected. Significant decrease in spermatogenesis was observed in each rat line, but the amount of decrease was reduced by resistance alleles. The highest TCDD dose decreased the daily sperm production by 37, 38, and 60% in line A, B, and C rats, respectively. Therefore, the resistance alleles appear to selectively modify the TCDD effects on the adult male reproductive system. The fact that the influence of resistance alleles on spermatogenesis is different from that on androgenic status indicates that the effect of TCDD on sperm numbers is not fully related to decreased serum testosterone.     

Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.     

Effects of in utero and lactational exposure to endosulfan in prefrontal cortex of male rats

The possible neurotoxic effects of the organochlorine pesticide endosulfan have been evaluated on male offspring rats exposed in utero and during lactation. Dams were treated with 0.61mg or 6.12mg endosulfan/(kgday) from the gestation beginning until the weaning. Male offspring rats were sacrificed at post-natal days (PND) 15, 30 and 60, and possible alterations in the content and metabolism of biogenic amines and amino acids were determined in prefrontal cortex using high-performance liquid chromatography (HPLC). Globally, endosulfan induced an increase in amino acid content in prefrontal cortex at PND 15 and PND 30. However, the levels of GABA at PND 15 and those of glutamine at PND 30 were not modified. At PND 60, a significant reduction in the content of GABA and taurine was observed, while the concentration of glutamate, aspartate and glutamine remained constant. Endosulfan did not modify norepinephrine and dopamine content, but serotonin concentration was increased at PND 30 and PND 60 and serotoninergic and dopaminergic metabolism was also modified. These results suggest that pre- and post-natal exposure to endosulfan affects biogenic amines and amino acids in prefrontal cortex, and those variations could be related to several alterations in the functions in which the prefrontal cortex is involved.     

In utero and lactational exposure to Aroclor 1254 affects bone geometry, mineral density and biomechanical properties of rat offspring

Exposure to polychlorinated biphenyls (PCBs) induce a broad spectrum of toxic effects in various organs including bone. The most susceptible age-groups to the toxic effects of PCBs are foetuses and infants. The aim of the present study was to quantitatively evaluate changes in bone geometry, mineral density and biomechanical properties following perinatal exposure to the PCB mixture, Aroclor 1254 (A1254), and to examine the persistence of observed bone alterations by following the offspring over time. Sprague-Dawley rat offspring were exposed to A1254 from gestational day 1 to post-natal day (PND) 23. Femur and tibia were collected on PNDs 35, 77 and 350 and were analyzed by peripheral quantitative computed tomography and biomechanical testing. At PND35, exposure to A1254 induced short, thin femur and tibia, with reduced mechanical strength of femoral neck. No treatment-related bone changes were detected in offspring at PND77 or PND350. In conclusion, the present investigation suggests that perinatal exposure to A1254 leads to shorter, thinner and weaker bones in juvenile rats at PND35, with these effects being absent at later time-points as exposure is discontinued. The results indicate that the observed bone effects are mainly driven by the dioxin-like congeners, although it cannot exclude the contribution of the non dioxin-like congeners to the exposure outcome.     

Evaluation of the persistence of hydronephrosis induced in mice following in utero and/or lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potent teratogen in mice, inducing structural malformations in the kidney and secondary palate. Maternal depots of TCDD, stored primarily in adipose tissue, are mobilized during the nursing period. Thus, lactation serves as a significant route of exposure for the developing neonate. The objective of this present investigation was to assess whether hydronephrosis persisted postnatally, as well as to determine if the renal lesion could be induced lactationally. Pregnant C57BL/6N mice were treated once by gavage with 0, 3, or 12 micrograms TCDD/kg body wt on Gestation Day (GD) 6. All dams were allowed to litter, and each litter was standardized at random to a size of six pups. Standardized litters were then reciprocally cross-fostered on the day of birth. Postnatal Day (PND) 0, resulting in the establishment of four experimental groups: pups not exposed by either route, pups exposed only in utero, pups exposed only lactationally, and pups exposed by both routes. Pups were euthanized at one of two time points, either at weaning (PND 25) or at puberty (PND 67). TCDD was not overtly toxic to the dams or neonates with the dosing regime used in this study. Hydronephrotic incidence and severity, while greatest for pups receiving dual exposure, were essentially the same for pups exposed in utero only vs lactationally only. Lactational exposure induced hydronephrosis (HN), as well as exacerbated the severity of existing HN which was induced in utero. Regardless of the exposure group, the severity of the renal lesion was always greater in the right kidney than in the left. There were no sex-related differences in either the incidence or the severity of HN, nor was there any difference in response between PNDs 25 and 67. These data suggest that the renal lesion persists from weaning through puberty, despite the cessation of exposure. However, the data indicate that partial recovery from HN induced in utero occurs during the early postnatal period, as both hydronephrotic incidence and severity decreased with increasing age between GD 18 and PND 25. Recovery was most pronounced in the left kidney regardless of dose, thus suggesting that the ability to recover may in part be dependent upon the extent of renal damage.     

Gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin affects social behaviors between developing rhesus monkeys (Macaca mulatta)

Pregnant rhesus monkeys (Macaca mulatta) were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 30 and 300 ng/kg by subcutaneous injection at gestational day 20, followed by additional injections of TCDD (1.5 and 15 ng/kg, respectively) every 30 days till 90 days after parturition. The offspring delivered from these experimentally TCDD-exposed mothers were subjected to a series of behavioral tests after the weaning at 12–14 months old (MO): a finger maze learning task (12–15 MO), encounter tests between two monkeys (at 12–15 and 24–27 MO), and an eye-contact test (23–26 MO) to estimate learning ability, social interaction with a peer subject, and interest or hostility to a human observer, respectively. TCDD exposure had no significant effect on learning ability or interest/hostility to an observer. It did, however, significantly affect behavioral characteristics in the encounter tests. In the first encounter test, monkeys exposed to TCDD showed more visual exploration and mutual proximity but less stereotypy behavior compared to control monkeys. In the second encounter test, these differences seemed to disappear, suggesting that the behavioral effects of TCDD exposure in the encounter tests might disappear as the monkey develops. This study produced evidence of the behavioral toxicity of TCDD in social interactions using non-human primates.     

Effects of maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on fetal brain growth and motor and behavioral development in offspring rats

The effects of maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy on fetal brain growth and neurobehavioral development in early developmental stages were investigated using rat offspring. TCDD in corn-oil (0.1microg/kg) was orally administrated to the dams from the 9th to 19th gestational day. When TCDD effects on the fetal brain weight were analyzed on the 19th gestational day, weight ratio of the brain to the whole body, and that of the forebrain without the cerebral cortex to the whole brain were larger in the exposed group than those of the control group, suggesting premature fetal brain development. TCDD effects on motor functions were investigated using newborns in an inclined plane task. Motor development assessed by righting response on an inclination was delayed in the exposed offspring in the 8th-12th postnatal day, especially in male. Also, TCDD effects on active avoidance behavior in a shuttle box were investigated using the offspring after weaning. Latency in the active avoidance learning was longer, and locomotor activity was reduced in the exposed male offspring in the 41st-44th postnatal day. The results demonstrated that maternal TCDD exposure delayed fetal brain growth and neurodevelopment of the offspring in early stage, especially in male rats.