Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens

We have isolated a human lymphoblastoid cell line with higher levels of native cytochrome P450IA1 activity and by DNA transfection introduced human cDNAs for a putative cytochrome P450IIA2 and epoxide hydrolase (E.C. 3.3.2.3). The resultant cell line, designated MCL-1, was substantially more sensitive to the mutagenicity of dimethylnitrosamine and benzo[a]pyrene than the AHH-1 cell line and was found to have increased metabolism of benzo[a]pyrene to dihydrodiols. The increase in native cytochrome P450IA1 activity was achieved by mutation and selection based on resistance to the phototoxicity of benzo[ghi]perylene. One resistant clone, designated L3, was used for subsequent studies. Two complete cDNAs, one encoding a putative cytochrome P450IIA2 and the other a microsomal epoxide hydrolase, were isolated from a human liver cDNA library. After introduction of the cDNAs into an expression vector and transfection into AHH-1 cells, gene expression was detected at the level of enzyme activity (epoxide hydrolase) or by increased sensitivity to dimethylnitrosamine cytotoxicity/mutagenicity (putative P450IIA2). A vector containing both cDNAs was then constructed and transfected into L3 cells to produce MCL-1 cells. The potential usefulness of drug-metabolizing gene transfection and of the MCL-1 cell line, in particular, for genetic toxicity testing is discussed.     

Misoprostol in preventing postpartum hemorrhage: a meta-analysis.

OBJECTIVE: To assess misoprostol's ability to prevent postpartum hemorrhage (PPH) where no alternatives exist. Comparison to oxytocics demonstrates how similarly misoprostol achieves a level of effectiveness-obtainable only in hospitals-in remote locations around the world. METHOD: Using the Mantel-Haenszel fixed-effects model and the DerSirmonian and Laird random-effects model, summary statistics indicated that misoprostol's excess risk of PPH was only 4% when compared to oxytocics. RESULT: This risk difference was well within the range of expected results for all uterotonic agents and does not warrant branding misoprostol as an inferior drug. CONCLUSION: Conventional uterotonic drugs should not be used to set the lowest-accepted level of effectiveness in settings where they are entirely unsuitable. Continuing to weigh the benefits of one effective drug against another only delays the distribution of misoprostol in countries where it is the only feasible choice and must be measured against no treatment at all.     

Tumor vascular signals in renal masses: detection with Doppler US

The vascularity of 49 renal masses (26 malignant and 23 benign lesions) was investigated with duplex Doppler ultrasound. Doppler signals obtained at the margins of renal masses were defined as "tumor signals" when the Doppler-shifted frequency of the lesion exceeded the frequency shift in the ipsilateral main renal artery. These exceeded 2.5 kHz with a 3-MHz insonating frequency. Among the 26 renal masses that subsequently proved to be malignant, tumor signals were obtained in 15 of 18 (83%) untreated renal cell carcinomas, in three of four Wilms tumors, and in two patients with metastases to the kidney, but not in the one patient with lymphoma. None of the 23 benign renal masses demonstrated tumor signals. Tumor vascularity in malignant lesions gives rise to abnormal, high-velocity, Doppler-shifted signals that can help in the differential diagnosis of renal masses.     

Susceptibility of cyclooxygenase-2-deficient mice to pulmonary fibrogenesis

The cyclooxygenase (COX)-2 enzyme has been implicated as an important mediator of pulmonary fibrosis. In this study, the lung fibrotic responses were investigated in COX-1 or COX-2-deficient (-/-) mice following vanadium pentoxide (V(2)O(5)) exposure. Lung histology was normal in saline-instilled wild-type and COX-deficient mice. COX-2(-/-), but not COX-1(-/-) or wild-type mice, exhibited severe inflammatory responses by 3 days following V(2)O(5) exposure and developed pulmonary fibrosis 2 weeks post-V(2)O(5) exposure. Western blot analysis and immunohistochemistry showed that COX-1 protein was present in type 2 epithelial cells, bronchial epithelial cells, and airway smooth muscle cells of saline or V(2)O(5)-exposed wild-type and COX-2(-/-) mice. COX-2 protein was present in Clara cells of wild-type and COX-1(-/-) terminal bronchioles and was strongly induced 24 hours after V(2)O(5) exposure. Prostaglandin (PG) E(2) levels in the bronchoalveolar lavage (BAL) fluid from wild-type and COX-1(-/-) mice were significantly up-regulated by V(2)O(5) exposure within 24 hours, whereas PGE(2) was not up-regulated in COX-2(-/-) BAL fluid. Tumor necrosis factor-alpha was elevated in the BAL fluid from all genotypes after V(2)O(5) exposure, but was significantly and chronically elevated in the BAL fluid from COX-2(-/-) mice above wild-type or COX-1(-/-) mice. These findings indicate that the COX-2 enzyme is protective against pulmonary fibrogenesis, and we suggest that COX-2 generation of PGE(2) is an important factor in resolving inflammation.     

Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.