The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.     

Experimental thromboembolic stroke in cynomolgus monkey

To develop an experimental model of thromboembolic stroke without intracranial surgery, an autologous blood clot was delivered to the middle cerebral artery (MCA) via the internal carotid artery in cynomolgus monkeys. Male cynomolgus monkeys, in which a chronic catheter had been earlier implanted in the left internal carotid artery, were used. The clot was flushed into the internal carotid artery under sevofluorane anesthesia. A neurologic deficit score was assigned after MCA embolization. After 24 h, cerebral infarct size and location were determined by the TTC staining method. Cerebral blood flow (CBF) was measured prior to and after MCA embolization, using positron emission tomography (PET). After embolization, long-lasting and profound extensor hypotonia of the contralateral upper and lower limbs, and mild to severe incoordination were observed. Contralateral hemiplegia was observed over the following 24 h. In gross morphologic observation of the brain, the lesions involved mostly the caudate nucleus, putamen, globus pallidus and insular cortex. CBF was maximally reduced in the left MCA territory, but not in the right MCA territory. This model is relevant to thromboembolic stroke in human in neurologic dysfunction and histopathologic brain damage.     

Delayed hemispheric neuronal loss in severely head-injured patients

Recent experimental studies have revealed that traumatic brain injury as well as ischemic brain injury can cause chronic progressive neuronal damage. In the present study, we demonstrate previously unreported delayed cerebral atrophy on computerized tomography (CT) scans in severely head-injured patients. Seventeen severely head-injured patients who required mild hypothermia to control intracranial hypertension after the failure of conventional therapies were retrospectively analyzed. All 17 patients survived more than 1 year. Delayed neuronal loss (DNL) was observed in only eight of the 17 patients. Eight patients with DNL required longer durations of mild hypothermia to control intracranial hypertension than nine patients without DNL. Six of these eight patients with DNL achieved functional recovery despite progressive atrophic changes demonstrated on CT scans. On CT scans, DNL was characterized by (1) the sudden appearance at several months postinjury of a low-density area in the hemisphere ipsilateral to the injury; (2) the preservation of essential cortical structure although related white matter structures showed severe atrophic changes; and (3) no spread of the low-density area to the contiguous territory of the other main cerebral artery. It is concluded that focal primary injury to underlying brain, if severe enough, can result in delayed hemispheric atrophy.     

Glomerular CD8+ cells predict progression of childhood IgA nephropathy

The aim of this study was to evaluate whether the infiltrating T-lymphocyte can be a predictor in the disease progression of IgA nephropathy (IgAN). Twenty children with IgAN, followed for more than 5 years, were divided into progressive (n=5) and non-progressive groups (n=15). We assessed glomerular and interstitial infiltration of T-lymphocytes (CD4⁺ and CD8⁺ cells) and expression of α-smooth muscle actin (α-SMA) and transforming growth factor- β (TGF- β) using an indirect immunofluorescence method on the renal biopsies. We analyzed their relationship to the degree of proteinuria, histological changes, and prognosis. The number of CD8⁺ cells in glomeruli and in interstitium was higher in the progressive group than in the non-progressive group. The glomerular α-SMA staining was more intensive in the progressive group than in the non-progressive group. Urinary protein and the degree of histological changes were also higher in the progressive group than in the non-progressive group. Among these markers, the number of glomerular CD8⁺ cells was the most apparent difference between the two groups. In conclusion, these results indicate that the number of glomerular CD8⁺ cells is the most sensitive predictor of disease progression in childhood IgAN. Received: 20 June 2000 / Revised: 7 February 2001 / Accepted: 8 February 2001     

Pleomorphic leiomyosarcoma: clinicopathologic and immunohistochemical study with special emphasis on its distinction from ordinary leiomyosarcoma and malignant fibrous histiocytoma

Pleomorphic leiomyosarcoma (PLMS) was recently described as a morphologic variant of leiomyosarcoma; however, its diagnostic criteria, as shown by morphologic features and biologic behavior, remain controversial. We describe 28 cases of pleomorphic sarcoma with pleomorphic areas in more than two thirds of the tumor and an ordinary leiomyosarcomatous fascicular area covering less than one third as PLMS. PLMS comprised 8.6% of all the leiomyosarcomas (322 cases) registered in our institute. Patients ranged in age from 31 to 89 years (average, 57.9 years). Seventeen patients (60.7%) were male and 11 were female. Tumor location was as follows: the extremities in 17 cases, the retroperitoneum or abdominal cavity in 7 cases, the chest/abdominal wall in 3 cases, and the scalp in 1 case. Histologically, all cases showed at least small foci of fascicles consisting of smooth muscle tumor cells, in addition to pleomorphic areas mimicking storiform-pleomorphic malignant fibrous histiocytoma. The border between pleomorphic and leiomyosarcomatous fascicular areas was sharp in 3 cases, gradual in 2 cases, and blending in 23 cases. Sixteen cases (57.1%) showed a typical storiform pattern, 6 cases revealed extensive stromal hyalinization, 6 cases showed a chronic inflammatory infiltrate, 2 cases had the foci of foamy xanthomatous cells, and 7 cases contained myxoid malignant fibrous histiocytoma-like areas covering less than 50% of the tumor. The tumors had a tendency to be of a morphologically higher grade (10 tumors were French Federation of Cancer Centers grade 2, 18 were grade 3). Five of 28 cases (18%) showed rhabdoid features. Immunohistochemically, all of the 28 tumors examined showed a positive reactivity for at least one smooth muscle marker (desmin, muscle-specific actin, and alpha-smooth muscle actin) in the leiomyosarcomatous fascicular areas. In the pleomorphic areas the expression of smooth muscle markers (desmin 10 of 28, muscle-specific actin 13 of 28, and alpha-smooth muscle actin 14 of 28) was significantly reduced, compared with that in leiomyosarcomatous fascicular area (desmin 18 of 28, muscle-specific actin 26 of 28, and alpha-smooth muscle actin 24 of 28). No significant difference was observed between the MIB-1 labeling index in the leiomyosarcomatous fascicular areas (26.10 on average) and that in the pleomorphic areas (26.17 on average). However, the MIB-1 labeling index in PLMS was significantly higher than that in ordinary leiomyosarcoma (n = 20, 12.86 on average) or storiform-pleomorphic malignant fibrous histiocytoma (n = 16, 16.63 on average). In 23 patients follow-up data were available with a duration of 1-239 months. Eleven patients developed metastases, and lung accounted for the most common site of metastasis (9 cases). Fifteen of 23 patients (65.2%) died of disease. Our results indicate that PLMS should be differentiated from ordinary leiomyosarcoma because of its high proliferative activities and rather aggressive biologic behavior.     

Unidimensional scale for dementia.

A cognitive test comprising 27 subscales was administered to 262 demented patients and 92 normal subjects. Principal factor analysis followed by varimax and Harris-Kaiser rotation and Guttman's scalogram analysis was performed. The analysis yielded three factors, i.e. "recent memory", "immediate memory or attention" and "remote memory". The relationships between the three-dimensional distribution of the scores and the DSM-IIIR grade of dementia indicated the existence of a continuum of dementia severity. Scalogram analysis showed unidimensionality in the difficulty level of the subscales as well as in the severity of the cases. Thus, the simple summary score can be used as a good measure of the severity of dementia.     

Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition

Thermodynamic and kinetic analyses of biomolecular interactions reveal details of the energetic and dynamic features of molecular recognition processes, and complement structural analyses of the free and complexed conformations. The recent improvements in both isothermal titration calorimetry and surface plasmon resonance sensoring provide powerful tools for analysing biomolecular interactions in thermodynamic and kinetic approaches. The thermodynamic and kinetic parameters obtained for binding between protein and DNA indicate the mechanism of specific DNA recognition, in the high-resolution structures of the protein-DNA complexes. The effects of temperature and ionic strength reflect the conformational changes of the protein and DNA molecules upon complex formation, including important contributions of water and solutes. When combined with mutational studies, the interactions can be reduced to several energetic contributions from individual contacts. These studies should be useful to determine general features of protein functions in genetic regulation.     

Use of 5-FU plus hyperbaric oxygen for treating malignant tumors: evaluation of antitumor effect and measurement of 5-FU in individual organs

PURPOSE: Hyperbaric oxygen (HBO) has been shown to increase tumor radiosensitivity. Several reports indicate that it also increases sensitivity to alkylating agents, but other reports suggest that it may speed angiogenesis and tumor growth. To throw light on these questions, we investigated the effects of HBO and 5-fluorouracil (5-FU), individually and in combination, on Sarcoma 180 implants in mice. METHODS: We administered 5-FU at a dose of 0.75 mg/mouse six times per week and HBO at 2 atm absolute pressure for 90 min six times per week, both 17 times in total. In combination treatment, HBO was administered immediately after 5-FU injection. RESULTS: Over the treatment period, tumor diameter increased 277.8% in the untreated control group, 244.1% in the group receiving HBO monotherapy, 182.7% in the group receiving 5-FU monotherapy, and 138.5% in the group receiving combination therapy. Concomitant HBO increased accumulation of 5-FU in the tumors, liver, and kidneys, but not in the brain, of recipient animals. CONCLUSIONS: Based on the above results, we conclude that concomitant HBO enhances the effects of 5-FU.