Gamma-irradiation induced apoptosis in peritoneal macrophages by oxidative stress. Implications of antioxidants in caspase mitochondrial pathway.

The in vivo and in vitro development of apoptosis induced by gamma-irradiation was studied in mouse peritoneal macrophages. The apoptosis index was measured by fluorescence microscopy and DNA electrophoresis. In vivo apoptosis was greatest eight days after 8 Gy total body gamma-irradiation. A DNA ladder electrophoretic pattern was only observed in the gamma-irradiated group. The participation of reactive oxygen species in apoptosis induction was investigated by pretreating mice with the antioxidants superoxide dismutase, catalase, vitamin E or lipopolysaccharide before gamma-irradiation. Measurement of serum lipoperoxides showed oxidative stress in the gamma-irradiated mice and the protection given by the antioxidants. These results were confirmed using in vitro cultures of peritoneal macrophages: gamma-irradiated groups and antioxidant-pretreated gamma-irradiation groups showed results similar to those observed with in vivo irradiation. A loss of mitochondrial membrane potential (delta psi(m)) was also observed by microscopy in the gamma-irradiated cell cultures. Experiments with caspase inhibitors confirmed the participation of caspase 3 and caspase 9.

Modification of the P-glycoprotein dependent pharmacokinetics of digoxin in rats by human recombinant interferon-alpha.

PURPOSE: This study was conducted to investigate in vivo the impact of interferon-alpha (IFN)-alpha on P-glycoprotein (P-gp) activity in rats by studying how its administration modifies the bioavailability of digoxin, a fairly pure P-gp substrate. METHODS: Human recombinant IFN-alpha was given to rats (n = 5-7 per group) daily for 8 days at different doses (IntronA) 10(6), 2.10(6), or 4.10(6) IU kg(-1), s.c.), whereas pegylated-IFN-alpha (ViraferonPeg), 29 microg kg(-1)) was given s.c. three times a week. Rats were then given digoxin (32 microg kg(-1)) i.v. or orally. The pharmacokinetics of digoxin was studied. Intestinal P-gp expression was also examined. RESULTS: The pharmacokinetics of i.v. administered digoxin was not modified by IFN-alpha, but a dose-dependent increase in areas under the curve (AUCs) was observed in the orally administered digoxin parameters in rats (AUCs: 392 +/- 83 min microg L(-1), p < 0.01 and 550 +/- 97 min microg L(-1), p < 0.001, respectively, vs. 286 +/- 111 min microg L(-1) for control). A decrease in P-gp expression in the ileum (relative intensities: 0.70 +/- 0.19 for 4 Million International Unit (MIU) kg(-1) IFN-alpha-treated animals vs. 1.00 +/- 0.13 for controls, p < 0.05) and mainly in the jejunum (relative intensities: 0.46 +/- 0.13 for 4 MIU kg(-1) IFN-alpha-treated animals vs. 1.00 +/- 0.08 for controls, p < 0.001) was observed. CONCLUSION: IFN-alpha induces in vivo a significant dose-dependent inhibitory effect on intestinal P-gp activity related to a local decrease in its expression, thereby predicting important clinical consequences when IFN-alpha and other P-gp substrates are associated.

In vivo adriamycin-induced apoptosis in peritoneal murine macrophages: partial participation of a caspase cascade.

BACKGROUND: Adriamycin (ADM) is a potent antitumor drug that induces apoptosis (AP) in tumor cells. AP is modulated by caspases and by mitogen-activated protein kinases (MAPK) as well as by the mitochondrial membrane potential (deltapsim). We studied the participation of these systems in peritoneal macrophages from ADM-treated mice. MATERIALS AND METHODS: Balb/c mice were either treated with ADM (5 mg/kg, i.p.) or with 0.85% NaCl solution (controls). One hour later, peritoneal cells were harvested and cultured for 28 h. AP was evaluated by ethidium bromide and acridine orange staining; deltapsim was monitored using a MitoCapture stain Kit; DNA integrity was assessed by electrophoretic analysis. Animals were treated (i.p.) 1 h before ADM administration with Z-LEHD-FMK, Z-DEVD-FMK, or Z-VAD-FMK (caspase-9, caspases-3, 7,10 and general caspase inhibitors, respectively) or with PD169316 (a MAPKp38 inhibitor). RESULTS: ADM induced a higher rate of AP and the characteristic electrophoretic DNA ladder pattern. Mice treated with caspases inhibitors plus ADM showed significant reductions in AP and DNA laddering; in contrast, no differences were observed in mice treated with PD169316 plus ADM in comparison with ADM alone. ADM also induced early loss of the deltapsim. CONCLUSION: In these experimental conditions, ADM induced AP in a mainly caspase-9-dependent manner and this was related to a reduction in the deltapsim.

Identification of DNA sequences and viral proteins of 6 human papillomavirus types in retinoblastoma tissue.

PURPOSE: To detect DNA and proteins of human papillomavirus (HPV) in paraffin-embedded retinoblastoma (RB) tissue, to identify the viral types present and to describe a possible association between the presence of HPV and a particular form of RB. MATERIALS AND METHODS: Fifty-one samples from ocular tissues of RB patients and of six controls enucleated for non-neoplastic reasons were obtained and analyzed by Polymerase Chain Reaction (PCR) with consensus primers to detect HPV. Viral type identification was performed by Restriction Fragment Length Polymorphisms (RFLP) analysis. To corroborate the presence of HPV, immunohistochemical analysis with a polyclonal anti-HPV antibody was performed in 10 RB cases and in all controls. RESULTS: Forty-two (82.3%) of the 51 samples were HPV-positive. HPV 6 was detected in 40 cases (95.2%), HPV 33 in 16 (38.1%), HPV 11 in 4 (9.5%) and HPV 31, 35 and 51 each in one case (2.3%). All controls were negative for HPV-DNA. The positive samples were PCR-tested for HPV 16 and 18 using specific primers, and were all negative. For immunohistochemical analysis, 7 out of 10 PCR-positive samples randomly chosen were positive; all six controls were negative. CONCLUSION: No differences in the HPV type distribution were found between the groups formed according to the tumor presentation or to the mode of inheritance.