Comparative effects of Mono-Butyl Phthalate in vitro on testis explants from the fetal rat and human: comparison with effects of Di-Butyl Phthalate in vivo in the rat

The incidence of disorders in human male reproductive health is increasing. It has been proposed this change is a direct consequence of environmental effects on growth and sex steroid induced alterations in programmed development. The aim of the experimental work described in this thesis was to investigate the effects of an ubiquitous environmental contaminant, the synthetic platiciser Di-butylphthalate (DBP) on the developing male reproductive tract. Any changes were compared to those symptoms of the human male clinical condition TDS (Testicular dysgenesis syndrome) which is increasing in incidence. To investigate whether DBP treatment induced any TDS-like changes, pregnant rats were gavaged daily with DBP at doses up to 500mg/kg/day from embryonic (e) day el3.5 up to e21.5. Morphological and hormonal parameters (testosterone, inhibin-B) were assessed in male rats aged e21.5 or adult ± in utero DBP exposure. Concurrently, untreated fetal rat and fetal human testis explants were cultured in vitro ± MBP (Mono butylphthalate), the DBP metabolite, at levels of up to 1 mM, to investigate whether the array of adverse effects seen with the in utero exposure could be induced in vitro. Hormone production (testosterone, inhibin-B) and explant morphology were compared ± MBP exposure. The in vitro experiments were restricted to 48h exposure duration so an additional in vivo treatment regime was established to compare the endpoints induced after just 48h exposure in utero. Pregnant rats were dosed daily by gavage with 500mg DBP/kg/day on el9.5 and e20.5 only. Morphological and hormonal parameters (testosterone, inhibin-B) were assessed in male rats aged e21.5 ± 48h in utero DBP exposure. Findings were compared against those from the original in utero studies ± 8 days DBP exposure.The long-term in utero exposure regime induced an array of changes in the phenotype of the male reproductive tract, evident in e21.5 and adult animals, including testis maldescent (cryptorchidism) and reduced fertility. Changes in testis morphology such as alteration in the distribution ofLeydig cells and abnormal nucleation of gonocytes at e21.5 were also seen. The production of testosterone in testes at e21.5 was also significantly reduced following the DBP treatment, including a significant reduction in the protein expression levels of the steroidogenic enzyme P450scc. The in vitro experiments were unable to show a significant decrease in testosterone production after 48h exposure to ImM MBP but when testosterone production was stimulated by hCG, the level of stimulation was significantly reduced when explants were co-incubated with hCG and ImM MBP. The adverse effect of MBP on hCG stimulated testosterone production was seen in media collected from both fetal rat and fetal human testis explants after 48h co-incubation. The short-term in utero exposure regime induced a generally less severe array of changes in the phenotype and testis architecture of the e21.5 male reproductive tract than those seen after 8-day in utero DBP exposure. However, a greater reduction in testis testosterone was seen than the long-term exposure induced, despite less of a reduction in the protein expression ofP450scc. The precise mechanism through which DBP induces its array of developmental abnormalities is still unclear but these studies support the hypothesis that even short term in utero exposure to DBP directly affects the developing testis, probably by acting on Leydig cells and disrupting normal testis endocrinolog

Effects of Monobutyl and Di(n-butyl) Phthalate in Vitro on Steroidogenesis and Leydig Cell Aggregation in Fetal Testis Explants from the Rat: Comparison with Effects in Vivo in the Fetal Rat and Neonatal Marmoset and in Vitro in the Human

BACKGROUND: Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. OBJECTIVES: Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. METHODS: Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15–19 weeks of gestation) were cultured for 24–48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. RESULTS: Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5–20.5) DBP treatment. In vitro, MBP (10(−3) M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10(−3) M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2–7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. CONCLUSIONS: These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro

Best practices for developmental toxicity assessment for classification and labeling.

Many chemicals are going through a hazard-based classification and labeling process in Europe. Because of the significant public health implications, the best science must be applied in assessing developmental toxicity data. The European Teratology Society and Health and Environmental Sciences Institute co-organized a workshop to consider best practices, including data quality and consistency, interpretation of developmental effects in the presence of maternal toxicity, human relevance of animal data, and limits of chemical classes. Recommendations included larger historical control databases, more pharmacokinetic studies in pregnant animals for dose setting and study interpretation, generation of mechanistic data to resolve questions about whether maternal toxicity is causative of developmental toxicity, and more rigorous specifications for what constitutes a chemical class. It is our hope that these recommendations will form the basis for subsequent consensus workshops and other scientific activities designed to improve the scientific robustness of data interpretation for classification and labeling

Best practices for developmental toxicity assessment for classification and labeling

Many chemicals are going through a hazard-based classification and labeling process in Europe. Because of the significant public health implications, the best science must be applied in assessing developmental toxicity data. The European Teratology Society and Health and Environmental Sciences Institute co-organized a workshop to consider best practices, including data quality and consistency, interpretation of developmental effects in the presence of maternal toxicity, human relevance of animal data, and limits of chemical classes. Recommendations included larger historical control databases, more pharmacokinetic studies in pregnant animals for dose setting and study interpretation, generation of mechanistic data to resolve questions about whether maternal toxicity is causative of developmental toxicity, and more rigorous specifications for what constitutes a chemical class. It is our hope that these recommendations will form the basis for subsequent consensus workshops and other scientific activities designed to improve the scientific robustness of data interpretation for classification and labeling

Best practices for developmental toxicity assessment for classification and labeling.

Many chemicals are going through a hazard-based classification and labeling process in Europe. Because of the significant public health implications, the best science must be applied in assessing developmental toxicity data. The European Teratology Society and Health and Environmental Sciences Institute co-organized a workshop to consider best practices, including data quality and consistency, interpretation of developmental effects in the presence of maternal toxicity, human relevance of animal data, and limits of chemical classes. Recommendations included larger historical control databases, more pharmacokinetic studies in pregnant animals for dose setting and study interpretation, generation of mechanistic data to resolve questions about whether maternal toxicity is causative of developmental toxicity, and more rigorous specifications for what constitutes a chemical class. It is our hope that these recommendations will form the basis for subsequent consensus workshops and other scientific activities designed to improve the scientific robustness of data interpretation for classification and labeling