Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells.

The Embryonic Stem Cell Test (EST) has been successfully validated as an in vitro method for detecting embryotoxicity, showing a good overall test accuracy of 78% [Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151-176]. Methylmercury was the only strong in vivo embryotoxicant falsely predicted as non-embryotoxic making the metal the most significant outlayer [Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the Embryonic Stem Cell Test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209-244]. The misclassification of methylmercury and the potential environmental exposure to developmental toxic heavy metals promoted our investigation of whether the EST applicability domain covers cadmium and arsenic compounds. The EST misclassified cadmium, arsenite and arsenate compounds as non-embryotoxic, even when including arsenic metabolites (methylarsonate, methylarsonous and dimethylarsinic). The reasons were the lack of higher cytotoxicity towards embryonic stem cells as compared to more mature cells (3T3 fibroblasts) or the absence of inhibition of cardiac differentiation by specific mechanisms rather than general cytotoxicity. Including EST data on heavy metals from the literature (lithium, methylmercury, trivalent chromium and hexavalent chromium) revealed that the test correctly predicted the embryotoxic potential of three out of the seven heavy metals, indicating an insufficient predictivity for such metals. Refinement of the EST prediction model and inclusion of additional toxicological endpoints could expand the applicability domain and enhance the predictive power of the test.

Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT).

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon-carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI)> or = 3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.

Embryotoxicity hazard assessment of methylmercury and chromium using embryonic stem cells.

The embryonic stem cell test (EST) has been scientifically validated (2001) as an in vitro embryotoxicity test, showing a good overall test accuracy of 78%. Methylmercury (MeHg) was the most significant outlayer identified, as the metal was the only strong in vivo embryotoxicant falsely predicted to be non-embryotoxic. The EST misclassification of MeHg, and the potential environmental exposure and developmental toxic hazards of heavy metals gave us the rationale to investigate whether the EST can correctly predict the embryotoxic potential of two heavy metals different from MeHg. The EST correctly classified trivalent chromium to be non-embryotoxic and hexavalent chromium to be embryotoxic, while we confirmed the misclassification of MeHg. MeHg causes developmental abnormalities in the brain. We therefore aimed to improve the in vitro prediction of MeHg embryotoxicity by including a neuronal ES cell differentiation assay. Differentiation of neuronal-like cells was demonstrated by real-time PCR experiments, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of nestin, neurofilament medium polypeptide, beta-tubulin III and microtubule-associated protein 2 (Mtap2) positive cells. We identified Mtap2 mRNA expression as a sensitive toxicological endpoint for MeHg-induced neuronal embryotoxicity, as Mtap2 mRNA was down-regulated in the presence of non-cytotoxic concentrations of MeHg. Noticeably, several other neuronal marker genes were unaffected by MeHg and Mtap2 expression was not affected until day 14 of differentiation. This implies that the total neuronal-like cell number was unchanged and that the down-regulation of Mtap2 expression reflects neuron-specific toxicity, i.e. instability of the neuron-specific microtubules, and arrest of the neuronal maturation. The fact, that most marker genes were unaffected by MeHg, stresses the importance of including an array of marker genes. In conclusion, our results imply that inclusion of additional target tissues and refinement of the current prediction model may enhance the predictive power of the EST.

Caries inhibition by and safety of Lactobacillus paracasei DSMZ16671.

Lactobacillus paracasei DSMZ16671, even if heat-killed, sensitively co-aggregates mutans streptococci specifically. Mutans streptococci are strongly implicated in caries induction in humans and rodents. We hypothesized: (1) that S. mutans recoveries from rats' teeth in vivo will decrease, with an associated decrease in caries, if these lactobacilli are fed to rats in an established caries model; and (2) that toxicological assays of these lactobacilli will show them to be benign. Four groups of specific-pathogen-free rats were formed: S. mutans 10449S-inoculated/16671-supplemented diet; un-inoculated/16671-supplemented diet; S. mutans 10449S-inoculated/placebo diet; and un-inoculated/placebo diet. Standard tests of toxicity and mutagenicity of heat-killed DSMZ16671 were performed. S. mutans recoveries were significantly reduced both in mid-experiment and at termination, as were caries lesion scores for the rats inoculated by S. mutans and fed the DSMZ16671 supplement, by comparison with controls. Neither toxicity nor mutagenicity of DSMZ16671 was detected. Use of heat-killed DSMZ16671 is efficacious in rats and appears safe.

Human monocytes express functional receptors for granulocyte colony-stimulating factor that mediate suppression of monokines and interferon-gamma.

In a double-blind, placebo-controlled, randomized study, 10 healthy men received either a single dose of 480 microg granulocyte colony-stimulating factor (G-CSF) or saline. Blood taken from the volunteers was stimulated with 10 microg/mL endotoxin and released cytokines were measured by enzyme-linked immunosorbent assay. Expression of G-CSF receptors on leukocytes was examined by flow cytometry and reverse transcriptase-polymerase chain reaction. Functional activity of these receptors was tested by challenging isolated leukocyte populations to release cytokines with endotoxin in the presence of G-CSF. The G-CSF treatment attenuated the release of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-12, IL-1beta, and interferon (IFN)-gamma in ex vivo lipopolysaccharide (LPS)-stimulated whole blood. In blood from untreated volunteers the presence of G-CSF in vitro also attenuated the LPS-stimulated release of these cytokines. G-CSF in vitro also attenuated TNF-alpha release from elutriation-purified monocytes. In the presence of 10 ng/mL recombinant TNF-alpha, the attenuation of LPS-inducible IFN-gamma release by G-CSF was blunted in whole blood. However, G-CSF had no such effect on IFN-gamma release from isolated lymphocytes stimulated with anti-CD3 or a combination of TNF-alpha and IL-12. G-CSF receptor expression was detected in human neutrophils and monocytes but not in lymphocytes by means of RT-PCR as well as flow cytometry. These results indicate that G-CSF receptors expressed on monocytes are functional in modulating monokine release. We conclude that the attenuation of IFN-gamma release from lymphocytes is not a direct effect of G-CSF on these cells but is rather due to the inhibition of monocytic IL-12 and TNF-alpha release by G-CSF. (Blood. 2000;95:270-276)