Cadmium Impairs p53 Activity in HepG2 Cells.

Cadmium and cadmium compounds are contaminants of the environment, food, and drinking water and are important constituents of cigarette smoke. Cd exposure has also been associated with airborne particulate CdO and with Cd-containing quantum dots in medical therapy. Adverse cadmium effects reported in the literature have stimulated during recent years an ongoing discussion to better elucidate cadmium outcomes at cell and molecular level. The present work is designed to gain an insight into the mechanism of p53 impairment at gene and protein level to understand Cd-induced resistance to apoptosis. We used a hepatoma cell line (HepG2) derived from liver, known to be metal responsive. At genotoxic cadmium concentrations no cell cycle arrest was observed. The p53 at gene and protein level was not regulated. Fluorescence images showed that p53 was correctly translocated into the nucleus but that the p21(Cip1/WAF-1), a downstream protein of p53 network involved in cell cycle regulation, was not activated at the highest cadmium concentrations used. The miRNAs analysis revealed an upregulation of mir-372, an miRNA able to affect p21(Cip1/WAF-1) expression and promote cell cycle progression and proliferation. The role of metallothioneins and possible conformational changes of p53 are discussed.

Selective inhibition of B lymphocytes in TBTC-treated human bone marrow long-term culture.

Tributyltin chloride (TBTC) is well known for its immunotoxic effect, in particular towards immature thymocytes. TBTC is also known to induce adipocyte differentiation in primary human bone marrow cultures, which is reflected in the decrease in a number of adipocyte-derived cytokines, chemokines and the adipocyte-linked hormone leptin. Since adipocytes influence haematopoiesis and lymphopoiesis for instance by these cytokines and hormones, we here investigated whether TBTC has an effect on specific lymphocyte subsets in human bone marrow primary cultures. FACS analysis showed a reduction of CD19/CD22-positive B cells by TBTC, both in the presence or absence of cytokines. The treatment did not cause a toxic effect on mature CD3+CD4+ and CD3+CD8+ T cells, suggesting selective TBTC toxicity on B lymphocytes in the presently used in vitro system.

Maintenance and characterization of lymphocytes in human long term bone marrow cultures to study immunotoxicity.

Immunotoxicity of xenobiotics is of growing concern for various levels in society, including industry and regulatory authorities. Despite that EU legislation aims at reducing the number of laboratory animals by promoting the development of alternative validated methods, at present, immunotoxicity is generally evaluated through standard in vivo toxicity studies. The lack of alternative methods is due, in particular, to the complexity of the immune system and its responses, but possibly alternative methods for immunosuppressive chemicals are most achievable. The present study describes a long term culture (LTC) method capable of inducing the formation of lymphocyte subsets from human mononuclear bone marrow cells that may allow evaluation of lymphotoxicity. The LTC consisted of a two stages: a myeloid stage to allow the formation of a stromal layer and a lymphoid stage to allow expansion of lymphocytes. Results show that the use of IL-7 in LTC inhibits precursor and mature B cells, while it supports the proliferation of CD3(+)CD8(+) and CD3(+)CD4(+) T-cells. The bone marrow LTC model may in future be used to test the effect of xenobiotics on stromal dependent lymphocyte formation.

TBTC induces adipocyte differentiation in human bone marrow long term culture.

Organotins are widely used in agriculture and the chemical industry, causing persistent and widespread pollution. Organotins may affect the brain, liver and immune system and eventually human health. Recently, it has been shown that tri-butyltin (TBT) interacts with nuclear receptors PPAR gamma (peroxisome proliferator-activated receptor gamma) and RXR (retinoid x receptor) leading to adipocyte differentiation in the 3T3 cell line. Since adipocytes are known to influence haematopoiesis, for instance through the expression of cytokines and adhesion molecules, it was considered of interest to further study the adipocyte-stimulating effect of TBTC in human bone marrow cultures. Nile Red spectrofluorimetric analysis showed a significant increase of adipocytes in TBTC-treated cultures after 14 days of long term culture. Real-time PCR and Western blot analysis confirmed the high expression of the specific adipocyte differentiation marker aP2 (adipocyte-specific fatty acid binding protein). PPAR gamma, but not RXR, mRNA was increased after 24 h and 48 h exposure. TBTC also induced a decrease in a number of chemokines, interleukins, and growth factors. Also the expression of leptin, a hormone involved in haematopoiesis, was down regulated by TBTC treatment. It therefore appears that TBTC induced adipocyte differentiation, whilst reducing a number of haematopoietic factors. This study indicates that TBTC may interfere in the haematopoietic process through an alteration of the stromal layer and cytokine homeostasis.

Induction of apoptosis and inhibition of telomerase activity in human bone marrow and HL-60 p53 null cells treated with anti-cancer drugs.

Telomerase plays a key role in the maintenance of chromosomal stability in tumours, and the ability of anti-cancer agents to inhibit telomerase activity is under investigation. In this study, we evaluated the effect of etoposide and taxol, on the telomerase activity and telomere length in human leukaemia p53 null cells and human bone marrow cells, as well as apoptosis and cell cycle modulation. Results showed that after exposure to the drugs, HL-60 cells as well as the human progenitors underwent a block in G2 and subsequently apoptosis, whereas stromal cells from bone marrow did not undergo a block in G2 or enter apoptosis after etoposide exposure. Telomere length increased in stromal cells after treatment with both etoposide and taxol whereas in HL-60 cells only after etoposide treatment with. Bax, bcl-2 and bcl-x change their expression in stromal cells, whereas bcl-x was induced after drug treatment and bcl-2 down regulated in progenitor cells. Our data suggest that telomerase activity and apoptosis are correlated and they seem to be modulated by a common gene, bcl-2.