I. Langonne

Assessment of the Developmental Toxicity, Metabolism, and Placental Transfer of Di-n-butyl Phthalate Administered to Pregnant Rats

The developmental toxicity and placental transfer of di-n-butyl phthalate (DBP) were evaluated in Sprague-Dawley rats given a single oral dose of DBP on Gestational Day 14. In the developmental toxicity study, dams were dosed with 0, 0.5, 1, 1.5, or 2 g DBP/kg and were necropsied on GD21. Increased incidence of resorptions and reduced fetal body weight were observed at 1.5 and 2 g/kg. Higher incidences of skeletal variations were found at doses > or = at 1 g/kg. No embryotoxic or teratogenic effects were observed at a dose of 0.5 g/kg. In the placental transfer study, dams were dosed with 0.5 or 1.5 g [14C]DBP/kg. Maternal and embryonic tissues were collected at intervals from 0.5 to 48 h. Embryonic tissues accounted for less than 0.12-0.15% of the administered dose. Levels of radiocarbon in placenta and embryo were one-third or less of those in maternal plasma. No accumulation of radioactivity was observed in the maternal or embryonic tissues. From HPLC analyses, it was shown that unchanged DBP and its metabolites mono-n-butyl phthalate (MBP) and MBP glucuronide were rapidly transferred to the embryonic tissues, where their levels were constantly lower than those in maternal plasma. MBP accounted for most of the radioactivity recovered in maternal plasma, placenta, and embryo. Unchanged DBP was found only in small amounts. These findings support the hypothesis that MBP, a potent teratogen, largely contributes to the embryotoxic effects of DBP.

Developmental toxicity of N-methyl-2-pyrrolidone administered orally to rats

The developmental toxicity of N-methyl-2-pyrrolidone (NMP) was studied in Sprague-Dawley rats after oral administration. Pregnant rats were given NMP at doses of 0 (distilled water), 125, 250, 500, and 750 mg/kg/day, by gavage, on gestational days (GD) 6 through 20. Significant decreases in maternal body weight gain and food consumption during treatment, and a reduction in absolute weight gain were observed at 500 and 750 mg/kg. The incidence of resorptions per litter was significantly higher than control at 500 mg/kg, and rose to 91% at 750 mg/kg. Examination of the foetuses revealed treatment-related malformations, including imperforate anus and absence of tail, anasarca, and malformations of the great vessels and of the cervical arches. The incidence of malformed foetuses per litter, and of litters with malformed foetuses was significantly increased at 500 and 750 mg/kg. At 250 mg/kg, one foetus showed malformations similar to those recorded at higher dosages. There was a dose-related decrease in foetal body weights (male, female, and total) that reached statistical significance at 250 mg/kg. A significant increase in incomplete ossification of skull bones and of sternebrae was also present at 500 and 750 mg/kg. In summary, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was 250 and 125 mg/kg/day, respectively. Thus, oral administration of NMP produced developmental toxicity below maternally toxic levels.

Developmental toxicity of trichloroethylene, tetrachloroethylene and four of their metabolites in rat whole embryo culture

The embryotoxicity of trichloroethylene (TRI) tetrachloroethylene (PER), and of four of their oxidative metabolites i.e. trichloroacetic acid, dichloroacetic acid, chloral hydrate, and trichloroacetyl chloride, was studied in vitro, using the rat whole embryo culture system. Embryos from Sprague-Dawley rats were explanted on gestational day 10 (plug day=day 0) and cultured for 46 h in the presence of the test chemical. All of the tested chemicals produced concentration-dependent decreases in growth and differentiation and increases in the incidence of morphologically abnormal embryos. TRI and PER produced qualitatively similar patterns of abnormalities, while TRI and/or PER metabolites, each elicited clearly distinguishable dysmorphogenic profiles. The presence of hepatic microsomal fractions in the culture medium produced marked decreases in TRI- and PER-induced embryotoxic effects, including mortality, severity of malformations, and delayed growth and differentiation.

Iron oxide particles modulate the ovalbumin-induced Th2 immune response in mice

This study was designed to investigate the modulatory effects of submicron and nanosized iron oxide (Fe(2)O(3)) particles on the ovalbumin (OVA)-induced immune Th2 response in BALB/c mice. Particles were intratracheally administered four times to mice before and during the OVA sensitization period. For each particle type, three different doses, namely 4×100, 4×250 or 4×500 μg/mouse, were used and for each dose, four groups of mice, i.e. group saline solution (1), OVA (2), particles (3), and OVA plus particles (4), were constituted. Mice exposed to OVA alone exhibited an allergic Th2-dominated response with a consistent increase in inflammatory scores, eosinophil numbers, specific IgE levels and IL-4 production. When the mice were exposed to OVA and to high and intermediate doses of iron oxide submicron- or nanoparticles, the OVA-induced allergic response was significantly inhibited, as evidenced by the decrease in eosinophil cell influx and specific IgE levels. However, the low dose (4×100 μg) of submicron particles had no significant effect on the OVA allergic response while the same dose of nanoparticles had an adjuvant effect on the Th2 response to OVA. In conclusion, these data demonstrate that the pulmonary immune response to OVA is a sensitive target for intratracheally instilled particles. Depending on the particle dose and size, the allergic response was suppressed or enhanced.

Effects of Mono‐n‐butyl Phthalate on the Development of Rat Embryos: In vivo and in vitro Observations

The present study was conducted to further characterize the embryotoxic effects mono-n-butyl phthalate, a major metabolite of the plasticizer di-n-butyl phthalate, and evaluate its role in the developmental toxicity of di-n-butyl phthalate. The embryotoxic effects of mono-n-butyl phthalate were compared to those of the parent compound di-n-butyl phthalate after a single oral administration of 1.8, 3.6, 5.4, or 7.2 mmol/kg di-n-butyl phthalate or mono-n-butyl phthalate to Sprague-Dawley rats on gestational day 10 (Day 10). Embryos were evaluated for growth and development on Day 12. Both chemicals induced concentration-dependent developmental toxicity (i.e. decreased growth and malformations) which became apparent at 3.6 mmol/kg. Di-n-butyl phthalate and mono-n-butyl phthalate were approximately equally potent and produced qualitatively similar dysmorphogenic effects. Macroscopically, the most common malformations involved the prosencephalon, the optic system, and the mandibular and maxillary processes. In addition, the embryotoxic potential of mono-n-butyl phthalate was evaluated in vitro using the rat whole embryo culture system. Day 10 embryos were cultured for 48 hr in the presence of 0.5 to 5 mM mono-n-butyl phthalate and were then evaluated as the embryos grown in utero. Mono-n-butyl phthalate was a potent direct acting embryotoxicant, causing concentration-related growth retardation and dysmorphogenesis. The spectrum of morphological defects observed in mono-n-butyl phthalate-exposed embryos in vitro was comparable to those seen in vivo in the embryos at the same developmental stage after maternal administration of di-n-butyl phthalate or mono-n-butyl phthalate. These data provide additional evidence in support of the hypothesis that mono-n-butyl phthalate may be the active species for the developmental toxicity of ingested di-n-butyl phthalate in rats.

EFFECT OF SUBMICRON AND NANO-IRON OXIDE PARTICLES ON PULMONARY IMMUNITY IN MICE

Due to advances in nanotechnology, exposure to particle compounds in the workplace has become unavoidable. Assessment of their toxicity on health is an important occupational safety issue. This study was conducted in mice to investigate the toxicological effects of submicron and nano-iron oxide particles on pulmonary immune defences. In that purpose, we explored for the first time, inflammatory and immune responses in lung-associated lymph nodes. For each particle type, mice received either a single intratracheal instillation at different concentrations (250, 375, or 500μg/mouse) or four repeated instillations at 500μg/mouse each. Cytokine production, inflammatory and innate immune response, and humoral immune response were respectively assessed 1, 2, and 6 days after particle exposures. Both types of particles induced lung inflammation associated with increased cytokine productions in lymph node cell cultures and decreased pulmonary immune responses against sheep erythrocytes. Natural killer activity was not modified by particles. In comparison to single instillation, repeated instillations resulted in a reduction of inflammatory cell numbers in both bronchoalveolar lavages and pulmonary parenchyma. Moreover, the single instillation model demonstrated that, for a same dose, nano-iron oxide particles produced higher levels of inflammation and immunodepression than their submicron-sized counterparts.

Assessment of the Developmental Toxicity and Placental Transfer of 1,2-Dichloroethane in Rats

This study evaluates the developmental toxicity and placental transfer of 1,2-dichloroethane (DCE) in rats. Sprague-Dawley rats were given 0-2.4 mmol DCE kg-1 day-1 by gavage, or were exposed for 6 hr per day to 0-300 ppm DCE by inhalation, from Day 6 to 20 of gestation. Maternal toxicity was observed after inhalation exposure to 300 ppm DCE and oral administration of 2.0 or 2.4 mmol DCE kg-1. There was no evidence of altered growth nor teratogenic effects after either inhalation or oral administration of DCE at any concentration tested. The time course disposition of 14C was examined over a 48-hr period in 12- and 18-day pregnant rats after a single oral dose of 1.6 mmol [14C]DCE kg-1. Peak concentrations of radiocarbon occurred between 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day 18) tissues accounted for 0.06 and 0.4% of the administered dose, respectively. Up to 4 hr, levels of radiocarbon in placenta and fetus were slightly less than in maternal plasma of 18-day pregnant rats and were two to five times higher at later periods. At 2 hr, unchanged DCE accounted for most of radioactivity (78-86%) recovered in maternal plasma, placenta, and fetus. Acidic metabolites and radioactivity bound to macromolecules increased up to 24 hr (0.01 mumol-eq DCE g-1) in either placental or fetal tissues. Thereafter, their levels declined more slowly than those in the maternal plasma. Results from this developmental toxicity study in rats confirm embryonic exposure to radiocarbon associated with [14C]DCE and/or its metabolites and has demonstrated the lack of observable teratogenic effects.

Modulation of acrylonitrile-induced embryotoxicity in vitro by glutathione depletion

The effects of glutathione (GSH) depletion on the embryotoxicity of acrylonitrile were assessed in vitro using the rat whole-embryo culture system. Day 10 rat embryos were cultured in rat serum medium for 6 h in the presence of 250 μMl-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of GSH synthesis, to deplete GSH in both embryo and visceral yolk sac. Following pretreatment, conceptuses were cultured for an additional 21 h in the presence of 152, 228, or 304 μM acrylonitrile. At the end of the culture period, conceptuses were assessed for survival, growth and development, malformations, and the protein and glutathione content of embryos and yolk sacs were assayed. Acrylonitrile alone produced concentrationrelated and statistically significant decreases in yolk sac diameter, crown-rump length, head length and number of somite pairs, as well as in embryonic and yolk sac proteins. The chemical also caused dysmorphogenesis of the brain and of the caudal extremity, and a concentration-related and statistically significant increase in GSH content in the yolk sac. Pretreatment with BSO significantly enhanced the embryotoxic effects of acrylonitrile. The conceptuses displayed further decreases in functional yolk sac circulation, yolk sac diameter, crown-rump and head length, when compared to either acrylonitrile or BSO alone. The incidence of caudal malformations and the severity of brain malformations produced by acrylonitrile were also increased. Marked decreases in embryonic and yolk sac GSH contents were observed after exposure to BSO alone or in combination with acrylonitrile. Thus, depletion in embryonic and yolk sac GSH by BSO enhanced teratogenic and growth retarding effects of acrylonitrile in vitro, suggesting that GSH plays a critical role in modulating acrylonitrileelicited embryotoxicity.

Difference in the developmental toxicity of ethylenethiourea and three N,N′-substituted thiourea derivatives in rats

Abstract Sprague-Dawley rats were administered ethylenethiourea (ETU), 1,3-dimethyl-2-thiourea (DMT), 1,3-dibutyl-2-thiourea (DBT), or 1,3-diphenyl-2-thiourea (DPT) by gavage from Days 6 to 20 of gestation. Daily dosage levels (mg/kg/day) were ETU at 0, 15, 25, and 35; DMT at 0, 15, 25, 50, 100, and 200; DBT at 0, 15, 25, 50, 100, and 200; and DPT at 0, 25, 50, 100, and 200. There was evidence of maternal toxicity at all doses of DMT and at doses ≥ 50 mg DBT/kg/day. DPT was embryolethal at 200 mg/kg/day. Fetotoxicity was observed at doses ≥ 15 mg DMT/kg/day, ≥ 15 mg DBT/kg/day, and ≥ 100 mg DPT/kg/day/ ETU was the only chemical tested that proved to be teratogenic.

Simultaneous analysis of the local and systemic immune responses in mice to study the occupational asthma mechanisms induced by chromium and platinum

As a result of industrial development, increased exposure to platinum and chromium compounds and the subsequent development of occupational asthma (OA) has been reported. Although specific IgE antibodies, an indicator of allergic asthma, against chromium and platinum have been detected in workers with OA, the immunopathological mechanisms involved in this disease are not fully understood. To better understand these complex mechanisms, the local and systemic immune responses were simultaneously analyzed in mice sensitized and challenged three, four, or five times with sodium hexachloroplatinate (Pt salt) and with potassium dichromate (Cr salt) via the respiratory route. Dinitrochlorobenzene (DNCB) and anhydride trimellitic (TMA) were included in this study as reference compounds that induce Th1 and Th2 responses respectively. All the compounds studied may provoke pulmonary sensitization. In the Pt salt-treated mice with a significant increase in local Th2 cytokine production, the increase in IgE and mucus production and in eosinophil number had a positive correlation with the number of challenges (r=0.942, 0.976, and 0.978 respectively), whereas in the Cr salt-treated mice with no local increase in Th2 cytokines, the increase in IgE production and eosinophil numbers had an inverse correlation with the number of challenges (r=-0.895 and -0.999 respectively). The Th2-dominated response induced by Pt salt was very close to that induced by TMA and may thrive after the fifth challenge, probably due to the constancy of the significant decrease in IFN-γ level in the spleens. The results of the present work may increase our understanding of the immunopathological mechanisms of OA induced by platinum and chromium, and emphasize the advantage of simultaneously analyzing local and systemic immune response when studying respiratory allergy.