Relative parameter sensitivity in prenatal toxicity studies with substances classified as developmental toxicants.

Developmental toxicity testing according to the globally standardized OECD 414 protocol is an important basis for decisions on classification and labeling of developmental toxicants in the European Union (EU). This test requires relatively large animal numbers, given that parental and offspring generations are involved. In vitro assay designs and systems biology paradigms are being developed to reduce animal use and to improve prediction of human hazard. Such approaches could benefit from the long-term experience with animal protocols and more specifically from information on the relevance of effects observed in these tests for developmental toxicity. Therefore, we have analyzed relative parameter sensitivity in 22 publicly available developmental toxicity studies, representing about one third of all classified developmental toxicants under European legislation. Maternal and fetal weight effects and fetal survival were most often affected parameters at the developmental Lowest Observed Adverse Effect Level (dLOAEL), followed by skeletal malformations. Specific end points such as cleft palate were observed in fewer studies at dLOAEL, but if observed may have been crucial in classification and labeling decisions. These results are similar to earlier studies using different selections of chemicals, indicating that in general classified developmental toxicants have a similar pattern of effects at the dLOAEL as chemicals in general. These findings are discussed within the perspective of the development of innovative alternative approaches to developmental hazard assessment.

Dose-dependent effects of soy phyto-oestrogen genistein on adipocytes: mechanisms of action.

The potential role of genistein in the prevention and treatment of obesity has attracted much attention among public and medical communities. Conversely, increasing evidence indicates that genistein as an endocrine-disrupting substance is likely to play a role in the aetiology of obesity. This review focuses on the role of soy phyto-oestrogen genistein in adipocytes and the underlying mechanisms of action. Genistein dose-dependently inhibits and stimulates adipogenesis in vitro. Increasing evidence shows that genistein dose-dependently influences obesity in both male and female animals. Dose-dependent effects of genistein on adipocytes vary with factors such as age and gender of animals. In addition, the role of developmental exposure of genistein in adult obesity has been discussed. Genistein, different from oestrogen, concurrently activates nuclear receptors, oestrogen receptors and peroxisome proliferator-activated receptors, and it inhibits various enzyme activities. The balance among these pleiotrophic effects of genistein determines its dose-dependent effects on adipocyte differentiation and function. Current data suggest that genistein could regulate adiposity. However, it remains uncertain whether genistein plays a beneficial role in the prevention and treatment of obesity. Additional evidence is required before firm conclusions showing that genistein decreases adiposity.

Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification.

AIMS/HYPOTHESIS: Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD). METHODS: Rats were exposed to an HFD or a low-fat diet (LFD). In vivo cardiac function was monitored by echocardiography. Substrate uptake and utilisation were determined in isolated cardiomyocytes. RESULTS: Feeding an HFD for 8 weeks induced left ventricular dilation in the systolic phase and decreased fractional shortening and the ejection fraction. Insulin-stimulated glucose uptake and proline-rich Akt substrate 40 phosphorylation were 41% (p < 0.001) and 45% (p < 0.05) lower, respectively, in cardiomyocytes from rats on the HFD. However, long-chain fatty acid (LCFA) uptake was 1.4-fold increased (p < 0.001) and LCFA esterification into triacylglycerols and phospholipids was increased 1.4- and 1.5-fold, respectively (both p < 0.05), in cardiomyocytes from HFD compared with LFD hearts. In the presence of the CD36 inhibitor sulfo-N-succinimidyloleate, LCFA uptake and esterification were similar in LFD and HFD cardiomyocytes. In HFD hearts CD36 was relocated to the sarcolemma, and basal phosphorylation of a mediator of CD36-trafficking, i.e. protein kinase B (PKB/Akt), was increased. CONCLUSIONS/INTERPRETATION: Feeding rats an HFD induced cardiac contractile dysfunction, which was accompanied by the relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The permanent presence of CD36 at the sarcolemma resulted in enhanced rates of LCFA uptake and myocardial triacylglycerol accumulation, and may contribute to the development of insulin resistance and diabetic cardiomyopathy.

Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway.

In vitro differentiation of the progenitor cells or preadipocytes into adipocytes is usually achieved by adding an adipogenic mixture (isobutylmethylxanthine, dexamethasone, and insulin, IDI) to medium supplemented with fetal bovine serum (FBS). To study the effects of steroid hormones in vitro, endogenous hormones, growth factors and cytokines are removed by charcoal stripping of serum. However, the effects of charcoal-stripped serum (CS-FBS) per se on adipogenesis have been ignored. Here, we showed that alkaline phosphate activity and nodule formation of osteoprogenitor KS483 cells were lower in CS-FBS than in FBS. Concurrently, abundant amounts of adipocytes were only observed in KS483 cells cultured with CS-FBS, irrespective of the brands of serum used. Inhibition of the p42/44 MAPK pathway by its specific inhibitor PD98059 increased adipogenesis of KS483 cells with FBS, whereas activation of this signalling pathway by EGF blocked adipogenesis of these cells with CS-FBS. Furthermore, the p42/44 MAPK phosphorylation of KS483 cells cultured with CS-FBS was decreased compared with FBS. We concluded that charcoal-stripping of serum removed stimulators of the MAPK signalling pathway and in turn led to downregulation of osteogenesis and upregulation of adipogenesis. Interestingly, the adipogenic mixture IDI stimulated adipogenesis of KS483 cells cultured with CS-FBS, but not with FBS. Furthermore, differential effects of genistein on adipogenesis were observed in KS483 cells cultured with FBS or CS-FBS in combination with IDI. Our results showed that charcoal stripping of serum affected the commitment of KS483 cells and therefore differentially regulated adipogenesis influenced by IDI alone and in combination with genistein.

Exposure of KS483 cells to estrogen enhances osteogenesis and inhibits adipogenesis.

Osteoblasts and adipocytes arise from a common progenitor cell in bone marrow. Whether estrogen directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. Using a mouse clonal cell line KS483 cultured in charcoal-stripped fetal bovine serum (FBS), we showed that 17beta-estradiol (E2) stimulates the differentiation of progenitor cells into osteoblasts and concurrently inhibits adipocyte formation in an estrogen receptor (ER)-dependent way. E2 increased alkaline phosphate (ALP) activity and nodule formation and stimulated messenger RNA (mRNA) expression of core-binding factor alpha-1 (Cbfa1), parathyroid hormone/parathyroid hormone-related protein receptors (PTH/PTHrP-Rs), and osteocalcin. In contrast, E2 decreased adipocyte numbers and down-regulated mRNA expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)2, adipocyte protein 2 (aP2), and lipoprotein lipase (LPL). Furthermore, the reciprocal control of osteoblast and adipocyte differentiation by E2 was observed also in the presence of the adipogenic mixture of isobutylmethylxanthine, dexamethasone, and insulin. Immunohistochemical staining showed that ERalpha and ERbeta were present in osteoblasts and adipocytes. A new mouse splice variant ERbeta2 was identified, which differed in two amino acid residues from the rat isoform. E2 down-regulated mRNA expression of ERalpha, ERbeta1, and ERbeta2. The effects of E2 are not restricted to the KS483 cell line because similar results were obtained in mouse bone marrow cell cultures. Our results indicate that estrogen, in addition to stimulation of osteogenesis, inhibits adipogenesis, which might explain the clinical observations that estrogen-deficiency leads to an increase in adipocytes.

Metallothionein and cortisol receptor expression in gills of Atlantic salmon, Salmo salar, exposed to dietary cadmium.

Commercial fish feeds may contain significant levels of cadmium (Cd). However, little is known about the effects of dietary cadmium on fish organs, especially gills, the key osmoregulatory organ. We therefore studied the effects of dietary cadmium on metallothionein (MT) and cortisol receptor (GR) immunoreactivity in the branchial epithelium of the Atlantic salmon (Salmo salar). Cadmium was daily administered via food at 0.2mg (control), 5mg (low dose) and 125 mg (high dose) Cd per kilogram dry pellet weight. Fish were sampled after four and eight weeks. After both four and eight weeks, plasma cadmium concentration had increased significantly only in fish fed the high cadmium dose. Plasma calcium, sodium, chloride and cortisol levels were not affected. In the controls, most MT was colocated with the chloride cell marker, Na(+)/K(+)-ATPase, but some MT was present in pavement and respiratory cells. GR expression was found in chloride, pavement, respiratory and undifferentiated cells in all fish groups, but cadmium accumulation and a marked stimulation of MT expression were seen only in the chloride cells in the gills of fish fed the high cadmium dose. Cadmium treatment did not alter GR expression. When the double staining technique for MT and GR was applied, a marked heterogeneity became apparent in the chloride, pavement and respiratory cells of both groups of cadmium-treated fish and in the control fish. Some fish showed double staining, others stained only for one of the antibodies, whereas other cells were negative for both. We conclude that cadmium entering the gut also enters the gills, where it accumulates in chloride cells and stimulates MT expression.