Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in Wistar rats.

The brominated flame retardant (BFR) hexabromocyclododecane was tested in a one-generation reproduction assay in Wistar rats, enhanced for endocrine parameters. A solution of the compound in corn oil was mixed in the feed, targeting at dietary exposure of 0-0.1-0.3-1-3-10-30-100 mg/kg body weight/day (mkd) in parental rats during 10 (males) or 2 (females) weeks premating, during gestation and lactation, and in their F1 offspring from weaning until final necropsy. Effects were assessed in F1 animals. Livers of these animals showed increased HBCD concentrations, in a dose-dependent way. The trabecular bone mineral density of the tibia was dose-dependently decreased in females (BenchMark Dose Lower confidence bound, BMDL=0.056 mkd). The IgG response after immunization with sheep red blood cells (SRBC) was increased in males (BMDL=0.46 mkd). Further sensitive effects were decreased weight of the testis (BMDL=1.5 mkd), increased fraction of neutrophilic granulocytes (BMDL=7.7 mkd), decreased concentration of apolar retinoids in female livers (BMDL=1.3 mkd), and decreased plasma alkaline phosphatase in females (BMDL=8.6 mkd). CYP19/aromatase activity in the ovary was correlated to the concentration of gamma-HBCD in the liver. A developmental origin of these effects is considered, and this is also true for sensitive effects observed in neurobehavioural testing in littermates from the same experiment, i.e. in the brainstem auditory evoked potentials and in a catalepsy test [Lilienthal, H., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G. Neurobehavioral effects of the brominated flame retardant hexabromocyclododecane (HBCD) in rats after pre- and postnatal exposure, in press]. The low BMDLs of these effects may raise concern for human health, particularly when based on body burdens of HBCD, which leads to critical margins of exposure particularly for the occupational setting.

A 28-day oral dose toxicity study in Wistar rats enhanced to detect endocrine effects of decabromodiphenyl ether (decaBDE).

Decabromodiphenyl ether (decaBDE) is a widely used brominated flame retardant, considered to be of low toxicity. However, previous toxicity studies applied exposure methods with low bioavailability of this compound, and the actual hazard of decaBDE for humans, which are environmentally exposed to decaBDE, may thus be underestimated in current risk assessments. The present 28 days oral toxicity study in Wistar rats was designed to facilitate detection of endocrine and immune modulating effects of decaBDE using an exposure protocol with improved bioavailability. A technical preparation of high purity decaBDE was thus tested by daily exposure through gavage with an emulsion of soy phospholipon/lutrol as a carrier. Most sensitive effect in males were increased weight of seminal vesicle/coagulation gland with BMDL of 0.2mg/kg bw/day and increased expression of hepatic CYP1A and CYP2B (BMDLs 0.5-0.7 mg/kg bw/day). In females the most sensitive effect was decreased activity of P450c17 (CYP17), which is a key enzyme in the androgen synthesis pathway, in adrenals (BMDL 0.18 mg/kg bw/day). These results suggest that decaBDE may represent an as yet unreported hazard for reproductive health.

A 28-day oral dose toxicity study enhanced to detect endocrine effects of a purified technical pentabromodiphenyl ether (pentaBDE) mixture in Wistar rats.

A 28-day subacute oral toxicity study was performed in Wistar rats with a purified preparation of the commercial pentabromodiphenyl ether (pentaBDE), DE-71. The applied OECD407 protocol was enhanced for endocrine and immune parameters, and to enable benchmark dose analysis. A vehicle control group and 7 dose groups were included, which received 0.27, 0.82, 2.47, 7.4, 22.2, 66.7 or 200 mg pentaBDE/kg bw/d (mkd). The liver appeared to be a key target organ, showing a marked increase of weight and centrilobular hepatocellular hypertrophy, probably due to the observed induction of P450 enzymes, notably CYP1A and CYP2B. A marked decrease of circulating total thyroxine (TT4) and an increase of plasma cholesterol were probably secondary to the liver effects. Furthermore, dose-dependently decreased weight of epididymis, seminal vesicles, and prostate, as well as sperm head deformities in males, and induction of CYP17 activity in adrenals in females were observed, all possibly related to anti-androgenic activity. Finally, we observed a substantial increase of large unstained cells in the blood and a decrease of apolar retinoids in the liver. All these effects had benchmark doses at the lower confidence bound (BMDL) in the low- or mid-dose range, but particular sensitive, potentially adverse effects were TT4 decrease (BMDLs 1.1 in males and 1.8 mkd in females), and decrease of hepatic apolar retinoids (BMDLs 0.5 mkd in males and 2.3 mkd in females). These results contribute to refinement of the hazard identification of pentaBDE and improved risk assessment of human exposure to this industrial chemical and environmental pollutant.

Endocrine effects of tetrabromobisphenol-A (TBBPA) in Wistar rats as tested in a one-generation reproduction study and a subacute toxicity study.

Endocrine effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) were studied in a one-generation reproduction assay in Wistar rats via repeated dietary exposure, applying eight dose groups at 0-3-10-30-100-300-1,000-3,000 mg/kg body weight/day (mkd). This design enables dose-response analysis and calculation of benchmark doses (BMDL). This reproduction study was preceded by a 28-day repeat dose subacute toxicity study, at 0-30-100-300 mkd. Major effects in the reproduction study included decreased circulating thyroxine (T4) with BMDLs of 31 (m) and 16 (f) mkd, and increased weight of testis and male pituitary (BMDLs of 0.5 and 0.6 mkd). The hypothyroxinemia correlated to a cluster of developmental parameters including delayed sexual development in females, decreased pup mortality, and effects on brainstem auditory evoked potentials [Lilienthal, H., Verwer, C.M., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G., 2008. Neurobehavioral effects of tetrabromobisphenol A (TBBPA) in rats after pre- and postnatal exposure. Toxicology]. A second cluster of parameters in F1 animals was correlated to increased testis weight, and included female gonad weight, endometrium height, CYP19/aromatase activity in the ovary, and plasma testosterone levels in males. These two correlation clusters suggest a dual action of TBBPA. The only effects in the subacute study were decreased circulating T4 and increased T3 levels in males (BMDLs 48 and 124mkd), and non-significant trends for these parameters in females, suggesting that the other effects in the reproduction study were induced during development. Combined with data of human exposure to environmental TBBPA, the margin of exposure for highly exposed populations can be calculated at 2.6, and current use of TBBPA may therefore be a matter of concern for human health.

A 28-day oral dose toxicity study enhanced to detect endocrine effects of hexabromocyclododecane in Wistar rats.

A 28-day repeated dose study in rats (OECD407) enhanced for endocrine and immune parameters was performed with hexabromocyclododecane (HBCD). Rats were exposed by daily gavage to HBCD dissolved in corn oil in 8 dose groups with doses ranging between 0 and 200 mg/kg bw per day (mkd). Evaluation consisted of dose-response analysis with calculation of a benchmark dose at the lower 95% one-sided confidence bound (BMDL) at predefined critical effect sizes (CESs) of 10-20%. The most remarkable findings were dose-related effects on the thyroid hormone axis, that is, decreased total thyroxin (TT4, BMDL 55.5 mkd at CES--10%), increased pituitary weight (29 mkd at 10%) and increased immunostaining of TSH in the pituitary, increased thyroid weight (1.6 mkd at 10%), and thyroid follicle cell activation. These effects were restricted to females. Female rats also showed increased absolute liver weights (22.9 mkd at 20%) and induction of T4-glucuronyl transferase (4.1 mkd at 10%), suggesting that aberrant metabolization of T4 triggers feedback activation of the thyroid hormone system. These effects were accompanied by possibly secondary effects, including increased cholesterol (7.4 mkd at 10%), increased tibial bone mineral density (> 49 mkd at 10%), both in females, and decreased splenocyte counts (0.3-6.3 mkd at 20%; only evaluated in males). Overall, female rats appeared to be more sensitive to HBCD than male rats, and an overall BMDL is proposed at 1.6 mkd, based on a 10% increase of the thyroid weight, which was the most sensitive parameter in the sequence of events.

Pertussis toxin-induced histamine sensitisation: an aspecific phenomenon independent from the nitric oxide system?

Mechanisms were studied initially to develop an in vitro safety test for detecting pertussis toxin toxicity in acellular pertussis vaccines based on the histamine sensitisation test. Maximal contractions and sensitivities to different agonists and adrenoceptor-induced contractions in Ca2+-free medium of isolated rat small mesenteric resistance arteries were significantly reduced by in vivo [30 microg/kg, intravenously (i.v.), day 5] or in vitro (10 microg/ml, 2 h) pertussis toxin pretreatment. Pertussis toxin-induced decrease in sensitivity of small mesenteric resistance arteries to noradrenaline was endothelium-dependent. Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM, 20 min) did not reestablish the sensitivity to noradrenaline. In vivo L-NAME treatment (0, 1, 10 or 30 mg/kg) of pertussis toxin-pretreated (15 microg/kg) rats did not reduce pertussis toxin-induced enhancement of the histamine-induced decrease in blood pressure and histamine (10, 30, 100 or 300 mg/kg) induced mortality. Finally, in vivo pertussis toxin pretreatment sensitises rats for sodium nitroprusside (50 microg/kg/min). We conclude that pertussis toxin-induced histamine sensitisation is caused by an interference of pertussis toxin with the contractile mechanisms of vascular smooth muscle of resistance arteries which indicates only an indirect role for histamine in the histamine sensitisation test.

In vivo pertussis toxin treatment reduces contraction of rat resistance arteries but not that of mouse trachea.

In order to develop an in vitro method for detecting residual pertussis toxin activity in acellular pertussis vaccines, the effects of in vivo pertussis toxin treatment on contraction and relaxation properties of isolated mouse trachea and of isolated rat small mesenteric resistance arteries were studied. In vivo pertussis toxin treatment (24 or 72 microg/kg, intraperitoneally (i.p.)) did not affect contraction and relaxation properties of isolated BALB/c or NIH mouse trachea. In vivo pertussis toxin treatment (30 microg/kg, intravenously) significantly reduced noradrenaline- or KCl-induced maximal contraction and reduced sensitivity to noradrenaline in isolated male Wistar rat small mesenteric resistance arteries. However, in vivo pertussis toxin treatment did not affect relaxation properties of isolated rat small mesenteric resistance arteries. These results support the hypothesis that vasoconstriction-regulating mechanisms and not airway constriction mechanisms are involved in pertussis toxin-induced histamine sensitisation. The vasoconstriction-regulating mechanisms may provide a lead for further development of an in vitro method for measuring biologically active pertussis toxin in acellular pertussis vaccines based on mechanisms involved in the histamine sensitisation test.

Pertussis toxin relaxes small arteries with no vascular lesions or vascular smooth muscle cell injury.

Previous studies showed that pertussis toxin (PT) decreased agonist-induced contractions of isolated rat small mesenteric resistance arteries independently from endothelium, nitric oxide-synthase or intracellular calcium concentrations. In this study, it was investigated if the PT-induced decreased contractile properties of small mesenteric resistance arteries could be a consequence of a PT-induced vascular and/or smooth muscle cell injury, leading to loss of contractile functionality. Male Wistar rats were treated with PT (30 microg/kg, intravenously) and sections of isolated small mesenteric resistance arteries were investigated with light- and electron microscopy. Light microscopic investigation of cross-sectioned small mesenteric resistance arteries of control animals clearly showed a contracted phase, while PT-pretreated animals showed a relaxed smooth inner surface of the vessel, indicating a vasodilated state. Electron microscopic investigation showed that PT-pretreatment neither induced vascular lesions nor caused morphological or numerical changes in cell organelles such as contractile elements of vascular smooth muscle cells. In conclusion, the PT-induced decreased contractile properties of isolated rat small resistance arteries are not caused by a PT-induced vascular and/or smooth muscle cell injury.