Evidence for distinct binding sites in the cumene hydroperoxide-dependent metabolism of benzo[a]pyrene catalyzed by cytochrome P-450

A few constitutive cytochrome P-450 isozymes in male rat liver microsomes catalyzed the metabolism of benzo[a]pyrene (BP) in cumene hydroperoxide (CHP)-dependent reactions, which produced predominantly 3-hydroxyBP and BP quinones. This process varied with the concentration of CHP. At 0.05 mM CHP, 3-hydroxyBP was the major metabolite. An increase in CHP concentration reduced 3-hydroxyBP formation but increased the level of BP quinones. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene selectively inhibited quinone formation and enhanced 3-hydroxyBP formation. Naphthalene, phenanthrene and benz[a]anthracene nonspecifically inhibited total metabolism. BP binding to microsomal protein correlated with quinone formation, suggesting a common precursor reactive intermediate. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less effective than that in the male. With females, quinones were the major metabolites at all CHP concentrations, and their formation was again modulated by pyrene. These data indicate that two distinct binding sites are responsible for the formation of 3-hydroxyBP and BP quinones.     

Surgical treatment of lymph nodes with metastatic melanoma from unknown primary site

To determine the prognosis of patients with lymph node metastases from an unknown primary melanoma, we retrospectively reviewed the clinicopathologic features of 188 such patients treated from 1971 through 1986 and compared their records with those of patients with clinical stage II melanoma with known primary lesions. Patients with lymph node metastases from an unknown primary melanoma represented 4.6% of all patients with melanoma treated during that period. The five- and ten-year survival rates were 42% and 40%, respectively (median, 37 months). When stratified by number of tumor-containing lymph nodes, there was no significant difference in survival between patients with an unknown primary melanoma and lymph node metastases and those with clinical stage II melanoma and known primary sites. The prognosis of the former patients is no worse than that of patients with lymph node metastases from a known primary site and should be treated in a comparable manner.     

Clinical experience with alpha-particle emitting 211At: treatment of recurrent brain tumor patients with 211At-labeled chimeric antitenascin monoclonal antibody 81C6.

alpha-Particle-emitting radionuclides, such as (211)At, with a 7.2-h half-life, may be optimally suited for the molecularly targeted radiotherapy of strategically sensitive tumor sites, such as those in the central nervous system. Because of the much shorter range and more potent cytotoxicity of alpha-particles than of beta-particles, (211)At-labeled agents may be ideal for the eradication of tumor cells remaining after surgical debulking of malignant brain tumors. The main goal of this study was to investigate the feasibility and safety of this approach in patients with recurrent malignant brain tumors. METHODS: Chimeric antitenascin monoclonal antibody 81C6 (ch81C6) (10 mg) was labeled with 71-347 MBq of (211)At by use of N-succinimidyl 3-[(211)At]astatobenzoate. Eighteen patients were treated with (211)At-labeled ch81C6 ((211)At-ch81C6) administered into a surgically created resection cavity (SCRC) and then with salvage chemotherapy. Serial gamma-camera imaging and blood sampling over 24 h were performed. RESULTS: A total of 96.7% +/- 3.6% (mean +/- SD) of (211)At decays occurred in the SCRC, and the mean blood-pool percentage injected dose was < or = 0.3. No patient experienced dose-limiting toxicity, and the maximum tolerated dose was not identified. Six patients experienced grade 2 neurotoxicity within 6 wk of (211)At-ch81C6 administration; this neurotoxicity resolved fully in all but 1 patient. No toxicities of grade 3 or higher were attributable to the treatment. No patient required repeat surgery for radionecrosis. The median survival times for all patients, those with glioblastoma multiforme, and those with anaplastic astrocytoma or oligodendroglioma were 54, 52, and 116 wk, respectively. CONCLUSION: This study provides proof of concept for regional targeted radiotherapy with (211)At-labeled molecules in oncology. Specifically, the regional administration of (211)At-ch81C6 is feasible, safe, and associated with a promising antitumor benefit in patients with malignant central nervous system tumors.     

Frozen section analysis of esophageal endoscopic mucosal resection specimens in the real-time management of Barrett's esophagus.

BACKGROUND & AIMS: The aim of this study was to assess the validity of frozen section analysis of endoscopic mucosal resection (EMR) specimens from Barrett's esophagus as compared with permanent sections for the detection of neoplasia. Frozen sections help to give immediate feedback for surgical procedures. It has not been determined whether EMR can be adequately interpreted by using frozen sections to aid endoscopists in completely resecting neoplastic lesions. METHODS: EMR specimens from Barrett's esophagus with high-grade dysplasia (HGD) and/or carcinoma were tested by frozen section. Pathologists evaluated EMR specimens for the depth of invasion as well as the appearance of clear margins of resection. The kappa statistic was calculated to assess the degree of agreement between the frozen section and permanent section diagnoses. RESULTS: Twenty-three consecutive patients underwent 30 EMRs with frozen section diagnosis. Frozen section revealed a carcinoma in 7 specimens (23%) and dysplasia in 20 (66%). Permanent sections found carcinoma in 8 specimens (26%), dysplasia in 19 specimens (63%), and normal or nondysplastic Barrett's esophagus in the remainder. The kappa statistic for the depth of invasion of EMR specimens was 0.93 (near perfect agreement). The kappa statistic for the margins of the EMR specimens was 0.80 (excellent agreement). CONCLUSIONS: This study indicated that frozen section analysis of esophageal EMR specimens is valid as compared with permanent section. This technique might allow rapid evaluation about the degree and depth of involvement of cancers. This allows physicians to make decisions regarding further therapy if margins are involved or decrease the use of EMR for histologically benign-appearing lesions.     

Effect of naringin on bone cells.

Statin, a HMG-CoA reductase inhibitor, was shown to increase BMP-2 gene expression for bone formation, by blocking the mevalonate pathway in cholesterol production. We investigated the effect of naringin, a flavonoid available commonly in citrus fruits, which was also a HMG-CoA reductase inhibitor, in UMR 106 osteoblastic cell line in vitro. The control group consisted of cells cultured without any intervention for different time intervals (24 h, 48 h, and 72 h), whereas the experimental (naringin) group consisted of cells cultured with naringin of different concentrations (0.001 micromol/L, 0.01 micromol/L, and 0.1 micromol/L) for the same time intervals of the control. Colorimetric Tetrazolium (MTT) assay, total protein content assay, and alkaline phosphatase activity were used to measure the cellular activities. Results for the naringin group showed an increase in MTT assay compared with the control and the effect was dose dependent. At high concentration (0.1 micromol), the increases ranged from 60% to 80%. In the total protein content assay, naringin also showed an increase compared with control and the effect was also dose dependent. At high concentration (0.1 micromol), the increases ranged from 9% to 20%. In the alkaline phosphatase activity assay, naringin at high concentration (0.1 micromol) significantly increased the activity up to 20%. In conclusion, naringin significantly increased bone cell activities in vitro. This is the first study specifically attempted to investigate the effect of naringin on bone cell activities. Besides statin, this provided another example of mevalonate pathway blockage in the cholesterol production pathway by HMG-CoA reductase inhibition will increase the bone cell activities.     

Radiosensitivity of mouse skin epithelial cell line established in serum-free culture: an alternative to animal use.

A cultured line of murine skin epithelial cells was established to investigate the potential use of cultured cells as an alternative to animal use in radiation research. C3Hf/Sed newborn mouse skin cells have been successfully cultured in serum- and Ca(2+)-free medium with no terminal differentiation to keratinized cells. Presently, more than 25 passages have passed with no loss of stem cell capability. The radiosensitivity and repair of sublethal and potentially lethal radiation damages were investigated in this epithelial cell line. The population cell doubling time was 25 +/- 2.9 hr at 37 degrees C. The clonal growth of epithelial cells after irradiation was performed in the serum-free medium in the presence of lethally irradiated skin fibroblasts. Single dose survival curves of exponentially growing epithelial cells were investigated from the seventh to the twenty-third passages, and no significant changes in radiosensitivity and doubling time were found. The confluent epithelial cells also showed an identical sensitivity to radiation. The alpha/beta ratios of survival curves fitted by the linear quadratic model were 6.1 +/- 1.0 and 5.9 +/- 1.3 Gy for cells in exponential and confluent phases, respectively. The survival curve of epithelial cells left in confluence for 8 hr after irradiation showed a smaller beta value than that of cells plated immediately after irradiation with a resultant alpha/beta ratio of 9.5 +/- 3.8 Gy. This alpha/beta ratio was identical to those found in many animal experiments, suggesting a potential use of this cell line as an alternative to animal use. The magnitude of repair of sublethal damage following 6 Gy was greater than that following 3.9 Gy. Survival curves were also obtained following twice-a-day irradiations with no sign of rapid repopulation. These results are discussed by comparing with published in vivo and in vitro data.     

APOE epsilon3 gene transfer attenuates brain damage after experimental stroke.

Apolipoprotein E (apoE, protein; APOE, gene) is the major lipid-transport protein in the brain and plays an important role in modulating the outcome and regenerative processes after acute brain injury. The aim of the present study was to determine if gene transfer of the epsilon3 form of APOE improves outcome in a murine model of transient focal cerebral ischaemia. Mice received an intrastriatal injection of vehicle, a second-generation adenoviral vector containing the green fluorescent protein gene (Ad-GFP) or a vector containing the APOE epsilon3 gene (Ad-APOE) 3 days before 60 mins focal ischaemia. Green fluorescent protein expression was observed in cells throughout the striatum and subcortical white matter indicating successful gene transfer and expression. ApoE levels in the brain were significantly increased after Ad-APOE compared with Ad-GFP or vehicle treatment. Ad-APOE treatment reduced the volume of ischaemic damage by 50% compared with Ad-GFP or vehicle treatment (13+/-3 versus 29+/-4 versus 27+/-5 mm(3)). The extent of postischaemic apoE immunoreactivity was enhanced in Ad-APOE compared with Ad-GFP or vehicle treated mice. These results show the ability of APOE gene transfer to markedly improve outcome after cerebral ischaemia and suggest that modulating apoE levels may be a potential strategy in human stroke therapy.