Fludarabine modulates expression of type VII collagen during haematopoietic stem cell transplantation for recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, complicated inherited blistering skin disease with few treatment options currently available. Recently, haematopoietic stem cell transplantation (HCT) has been used as an alternative therapy that can improve skin integrity, but it is not known if the preparative HCT regimen also contributes to the therapeutic response. OBJECTIVES: To determine whether chemotherapy drugs used in the HCT preparative regimen influence type VII collagen (C7) expression, which is inherently reduced or absent in RDEB skin, and to explore the pathomechanisms of such responses, if present. METHODS: Drugs from the HCT preparative regimen (busulfan, cyclophosphamide, ciclosporin A, fludarabine and mycophenolate) with inhibitors (PD98059, U0126, LY294002, SR11302, SIS3 and N-acetyl-l-cysteine) were added to normal human dermal and human RDEB fibroblasts. C7 expression was measured using reversetranscription polymerase chain reaction and immunoblotting. RESULTS: We uncovered a previously unknown consequence of fludarabine whereby dermal fibroblasts exposed to fludarabine upregulate C7. This effect is mediated, in part, through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B and transforming growth factor-ß pathways. Activation of these pathways leads to activation of downstream transcription factors, including activator protein 1 (AP-1) and SMAD. Subsequently, both AP-1 and SMAD bind the COL7A1 promoter and increase COL7A1 expression. CONCLUSIONS: Fludarabine influences the production of type VII collagen in RDEB fibroblasts.

Role of iron in ricin-induced lipid peroxidation and superoxide production.

Ricin has been shown to induce oxidative stress in the livers of mice in vivo. These studies examined ricin-induced hepatic microsomal lipid peroxidation in mice, and the modulation thereof by iron and desferrioxamine. In addition, the studies investigated the production of superoxide anion by microsomes, mitochondria, and macrophages. Ricin (25 micrograms/kg, in vivo) increased microsomal lipid peroxidation by approximately 1.8-fold relative to control animals. This effect was abrogated by adding desferrioxamine to the microsomes. Fe2+ increased lipid peroxidation approximately 15-fold and 5-fold when added to microsomes from control and ricin-treated animals, respectively. Adding ricin to microsomes from control animals, however, decreased lipid peroxidation in a concentration-dependent manner. Desferrioxamine decreased lipid peroxidation by 47% and 64% in the absence and presence of ricin (5 micrograms/ml), respectively. Ricin, added to mitochondria from untreated animals decreased lipid peroxidation by 26% and 17% in the presence and absence of Fe2+, respectively. The administration of ricin (5 and 25 micrograms/kg) to mice increased microsomal, mitochondrial and macrophage superoxide anion production, in a dose-dependent fashion. The results suggest that iron mediated production of superoxide anion may be involved in the process of oxidative stress induced by ricin.

Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats.

Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Recent studies have demonstrated that sodium dichromate [Cr(VI)] induces greater oxidative stress as compared with Cr(III), as indicated by the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes, and enhanced excretion of urinary lipid metabolites and hepatic DNA-single strand breaks (SSB) following acute oral administration of Cr(III) and Cr(VI). We have therefore examined the chronic effects of sodium dichromate dihydrate [Cr(VI); 10 mg (33.56 mumol)/kg/day] on hepatic mitochondrial and microsomal lipid peroxidation, enhanced excretion of urinary lipid metabolites including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), acetone (ACON) and propionaldehyde (PROP), and hepatic DNA damage over a period of 90 days. The maximal increases in hepatic lipid peroxidation and DNA damage were observed at approximately 45 days of treatment. Maximum increases in the urinary excretion of MDA, FA, ACT, ACON and PROP were 3.2-, 2.6-, 4.1-, 3.3- and 2.1-fold, respectively, while a 5.2-fold increase in DNA-SSB was observed. The results clearly indicate that chronic sodium dichromate administration induces oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of hexavalent chromium.

The modulating effects of tumor necrosis factor alpha antibody on ricin-induced oxidative stress in mice.

Previous studies in our laboratory have shown that the protein toxin ricin induces an oxidative stress in mice, resulting in increased urinary excretion of malondialdehyde (MDA), formaldehyde (FA), and acetone (ACON). Other toxicants have been shown to induce oxidative stress by macrophage activation with subsequent release of reactive oxygen species and tumor necrosis factor alpha (TNF-alpha). Therefore, the ability of TNF-alpha antibody to modulate ricin-induced urinary carbonyl excretion as well as hepatic lipid peroxidation, glutathione depletion, and DNA single-strand breaks was assessed. Ricin-induced urinary MDA, FA, and ACON were reduced significantly in mice receiving antibody (15,000 U/kg) 2 hours before treatment with ricin (5 micrograms/kg). At 48 hours following ricin treatment, MDA, FA, and ACON concentrations in the urine of TNF antibody-treated mice decreased 25.7, 53.2, and 64.5%, respectively, relative to ricin-treated mice receiving no antibody. In addition, anti-TNF-alpha (1500 U/kg) significantly decreased hepatic lipid peroxidation and DNA single-strand breaks, induced by 5 micrograms ricin/kg, by 49.3 and 44.2%, respectively. The results suggest that macrophage activation and subsequent release of TNF-alpha are involved in ricin toxicity.

Modulation of ricin toxicity in mice by biologically active substances.

Ricin is a highly toxic protein produced by the castor bean (Ricinus communis). It is one of various protein toxins that consist of two subunits joined by a disulfide bridge. One chain facilitates entry of the toxin into the cell while the other chain exhibits RNA N-glycosidase activity, which attacks a specific site on 28S rRNA, preventing polypeptide elongation and leading to cell death. Although ricin and other protein toxins are potential health hazards, no antidote against these toxins exists. Thus, a number of selected compounds were screened for their ability to alter ricin lethality in mice, based on percentage survival and time to death following a ricin LD100 of 25 micrograms kg-1 i.p. While no compound tested prevented lethality, dexamethasone and difluoromethylornithine (DFMO) significantly extended survival time. The effects of DFMO on ricin toxicity were markedly influenced by altering various pharmacokinetic parameters. The antioxidants butylated hydroxyanisole and vitamin E succinate also extended survival time in response to a lethal dose of ricin, but to a lesser extent than did dexamethasone and DFMO. The Golgi apparatus inhibitors monensin, swainsonine and tunicamycin enhanced ricin toxicity, as evidenced by shortened survival times. In addition, various nucleoside analogs, including acyclovir and trifluridine as well as adenosine, guanosine and dibutyryl cyclic AMP, also potentiated the toxicity of ricin. The results demonstrate that the toxicity of ricin is modulated by a wide variety of structurally distinct chemicals and may involve different mechanisms. Furthermore, the extent and direction of the modulation of ricin toxicity is highly dependent upon pharmacokinetic variables, including dose and dosing interval.

Ricin-induced hepatic lipid peroxidation, glutathione depletion, and DNA single-strand breaks in mice.

The ability of ricin, the glycoprotein toxin from the castor bean (Ricinus communis), to stimulate oxidative stress was investigated. Following the i.p. administration of 25 micrograms ricin/kg or the vehicle to female CF-1 mice, the effects of ricin on hepatic lipid peroxidation, nonprotein sulfhydryl (reduced glutathione) content and DNA single-strand breaks were determined at 0, 12, 24, 36, 48 and 72 hr post-treatment. Hepatic lipid peroxidation significantly increased 3.4-, 3.8-, and 3.0-fold relative to control values at 24, 36, and 48 hr post-treatment, respectively. Hepatic nonprotein sulfhydryl concentrations decreased significantly to approximately 51%, 61% and 65% of control values, at 24, 36, and 48 hr, respectively. The incidence of hepatic DNA single-strand breaks increased by 2.9-, 2.8-, and 2.4-fold relative to the zero time values at 24, 36, and 48 hr after treatment with ricin, respectively. No significant differences were observed in either lipid peroxidation or nonprotein sulfhydryl concentrations at 12 or 27 hr post-treatment. Decreases in liver and intestinal weight to body weight ratios were observed in ricin-treated animals, while no changes were observed in spleen and kidney weight to body weight ratios. These results indicate that in the liver of mice, ricin induces an oxidative stress which is maximal at approximately 36 hr post-treatment.