Expression and function of cytochrome p450 in brain drug metabolism.

Cytochrome P450 (CYP, P450) is the collective term for a superfamily of heme-containing membrane proteins responsible for the metabolism of approximately 70 - 80 % of clinically used drugs. Besides the liver and other peripheral organs, P450 isoforms are expressed in glial cells and neurons of the brain. To enlighten their function and significance is a topic of high interest, as most of the neuroactive drugs used in therapy today are not only substrates, but also inducers of brain P450s with far reaching consequences. First of all, brain P450s are regulated differentially from those in liver. The availability of the prosthetic heme group appears to be essential for correct membrane insertion and enzymatic functionality of brain P450s. Furthermore, although not contributing to body's overall drug metabolism, brain P450s fulfil particular functions within specific cell types of the brain. In astrocytes of brain's border lines P450 isoforms are expressed at very high level. They form a metabolic barrier regulating drugs' influx, modulate blood-flow regulation, and act as signalling enzymes in inflammation. In neurons, however, P450s apparently have different function. In specified brain regions such as hypothalamus, hippocampus and striatum they provide signalling molecules like steroids and fatty acids necessary for neuronal outgrowth and maintenance. Induction of these P450s by neuroactive drugs can alter steroid hormone signalling directly in drug target cells, which may cause clinically relevant side effects like reproductive disorders and sexual or mental dysfunction. The understanding of brain P450 function appears to be of major interest in long-term drug mediated therapy of neurological diseases.

Nodular intercapillary glomeruloscerosis in diabetes secondary to chronic calcific pancreatitis.

Nodular (specific) intercapillary glomerulosclerosis (Kimmelstiel-Wilson) was found at autopsy in a 47-year-old man who had been diabetic for 20 years. The family history for this disease had been negative. Both the clinical course and the autopsy findings strongly suggest that this patient's diabetes was secondary to chronic fibrocalcific pancreatitis. This is only the fourth recorded case of histologically documented nodular glomerulosclerosis occurring in a patient with pancreatogenic diabetes.

Role of T cell receptor delta gene in susceptibility to celiac disease.

There is a strong genetic influence on the susceptibility to celiac disease. Although in the vast majority of patients with celiac disease, the HLA-DQ(alpha1*0501, beta1*0201) heterodimer encoded by the alleles HLA-DQA1*0501 and HLA-DQB1*0201 seems to confer the primary disease susceptibility, it cannot be excluded that other genes contribute to disease susceptibility, as indicated by the difference in concordance rates between monozygotic twins and HLA identical siblings (70% vs. 30%). Obviously other genes involved in the genetic control of T cell mediated immune response could potentially influence susceptibility to celiac disease. The density of T cells using the gammadelta T cell receptor (TCR) is considerably increased in the jejunal epithelium of patients with celiac disease, an abnormality considered to be specific for celiac disease. This suggests an involvement of gammadelta T cells in the pathogenesis of the disease. To ascertain whether the TCR delta (TCRD) gene contributes to celiac disease susceptibility we carried out an association study and genetic linkage analysis using a highly polymorphic microsatellite marker at the TCRD locus on chromosome 14q11.2. The association study demonstrated no significant difference in allele frequencies of the TCRD gene marker between celiac disease patients and controls; accordingly, the relative risk estimates did not reach the level of statistical significance. In the linkage analysis, performed in 23 families, the logarithm of the odds (LOD) scores calculated for celiac disease versus the TCRD gene marker excluded linkage, suggesting that there is no determinant contributing to celiac disease status at or 5 cM distant to the analyzed TCRD gene marker. In conclusion, the results of the present study provide no evidence that the analyzed TCRD gene contributes substantially to celiac disease susceptibility.

Effect of resistance training on resting metabolic rate and its estimation by a dual-energy X-ray absorptiometry metabolic map.

BACKGROUND/OBJECTIVES: Fat-free mass (FFM) is the major predictor of resting metabolic rate (RMR). As protein supplementation during resistance training may augment gains in FFM, we investigated the effects of resistance training combined with protein supplementation on RMR and whether RMR responses could be estimated by a dual-energy X-ray absorptiometry (DXA) metabolic map. SUBJECTS/METHODS: Healthy adults completed a whole-body periodized resistance training program consisting of 96 workouts (~9 months). Participants were randomly assigned to supplement with whey protein (whey; n=18), soy protein (soy; n=21) or carbohydrate (carb; n=22). RMR was measured using indirect calorimetry (RMR(IC)) and estimated by DXA metabolic mapping (RMR(MM)) pretraining and posttraining. RESULTS: RMR(IC) increased from pretraining to posttraining in the whole cohort (1653±302 to 1726±291 kcal/day, P=0.001) without differences between the groups. Delta RMR(IC) and RMR(MM) (73±158 vs 52±41 kcal/day were not significantly different by t-test (P=0.303), although they were not significantly correlated (r=0.081; P=0.535). Stepwise regression identified 43% of the shared variance in delta RMR(IC) using total serum thyroxine, RMR(IC) and FFM at baseline (P=0.009). CONCLUSIONS: These results indicate that 9 months of resistance training significantly increased RMR ~5% on average, but there was wide variability between individuals, which can be partially accounted for by changes in FFM and thyroid hormones.

Distinct expression patterns and levels of enzymatic activity of matrix metalloproteinases and their inhibitors in primary brain tumors.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been implicated in the immense invasive potential and neovascularization of primary brain tumors. We investigated the gene expression profiles of MMPs 1, 2, 3, 7, 9, 12, 13, 14, 16 and of TIMPs 1, 2, 3, and 4 in various primary brain tumors (astrocytoma WHO grade I-III, glioblastoma, PNET, ependymoma III and oligoastrocytoma II) using novel RNase protection assay probe sets. In addition, we determined the level and cellular source of gelatinolytic activity and localized gelatinase B and TIMP-1 RNA. Distinct expression patterns of the MMP and TIMP genes were found in the various brain tumors tested. While the WHO grade I and II tumors had MT1/MT3 ratios below 1, the malignant (grade III and IV) tumors had ratios above 1. Strong expression of TIMP-1 RNA was observed in all malignant tumors and in grade I pilocytic astrocytomas and localized to the walls of neovessels. Quantitative analysis of enzymatic activity in the soluble fraction of protein extracts revealed that in most tumors gelatinases remained in the inactive pro-form. In situ zymography revealed net gelatinolytic activity in neurons of normal brain and in tumor cells and vessel walls of all tumors tested. Immunohistochemistry demonstrated that gelatinase B was localized to vessel walls, to neutrophils in areas of hemorrhage, and in glioblastomas to macrophages. Together these data demonstrate that the different primary brain tumors show distinct regulation of MMP and TIMP genes. The localization of the soluble gelatinase B indicates an association with neovascularization, whereas membrane-bound MMPs may account for the invasive potential of the glial tumor cells.

Molecular genetic analysis as a tool for evaluating stereotactic biopsies of glioma specimens.

Over the last years, distinct genetic lesions have been associated with individual tumor entities. Stereotactic biopsy has become an essential diagnostic tool in surgical neuro-oncology. In order to evaluate the potential of molecular analyses in stereotactic biopsies, we examined a series of 156 human brain tumors from patients undergoing stereotactic biopsy for molecular alterations typically seen in astrocytic gliomas and compared those results with a control group of 268 astrocytic tumors obtained at open surgery. Stereotactic biopsies of astrocytomas with borderline histopathological features between the WHO grades II and III showed a higher rate of allelic losses on chromosome 10 than those of the WHO grade II from open surgery (p = 0.011). Stereotactic biopsies of astrocytomas with borderline histopathological features between the WHO grades III and IV showed a higher rate of allelic losses on chromosome 10 than those of the WHO grade III from open surgery (p = 0.013). This indicates that stereotactic biopsies with features intermediate between grades are likely to correspond to the higher malignancy grade. Our data demonstrate that molecular genetic approaches can be successfully applied to stereotactic glioma biopsies. The difference in the distribution of malignancy associated genetic alterations between a stereotactic and openly resected group of gliomas indicates that histopathology may underestimate the malignant potential in some stereotactic specimens. We propose to further evaluate the molecular analysis of stereotactic glioma biopsies as a useful adjunct to standard histopathological procedures.

Chronic alcohol consumption induces lipofuscin deposition in the rat hippocampus.

Previous investigations have suggested that chronic alcohol consumption accelerates a number of age-related changes in the cerebellar cortex and hippocampal formation. In the cerebellum, alcohol-feeding has been shown to accelerate the intracellular deposition of lipofuscin. In order to determine whether alcohol administration has a similar effect on hippocampal lipofuscin deposition, we studied the pattern of lipofuscin deposition in alcohol-fed rats for periods of 1, 3, 6, 12 and 18 months and compared the results with those obtained in the respective pair-fed controls. A precocious and progressive deposition of lipofuscin pigment was found in both CA1 and CA3 neurons in Ammon's horn hippocampal fields after 3 and 6 months of alcohol feeding, respectively. These results parallel those observed during normal aging and reinforce the hypothesis of a close link between chronic alcohol consumption and a premature nerve cell aging.

Interleukin-6-associated inflammatory processes in Alzheimer's disease: new therapeutic options.

The cytokine interleukin-6 is consistently detected in the brains of Alzheimer's disease patients but not in the brains of nondemented elderly persons. Until recently it was unclear whether an interleukin-6-associated inflammatory mechanism is an early or late event in the pathological cascade of Alzheimer's disease. We investigated whether interleukin-6 could be detected in plaques of Alzheimer's disease patients prior to the onset of neuritic degeneration. We found interleukin-6 mostly in plaques where neuritic pathology has not yet developed. This indicates that the appearance of interleukin-6 may precede neuritic changes and is not just a consequence of neuritic degeneration. Therefore, one may hypothesize that activation of inflammatory mechanisms may cause neuritic degeneration in plaques. A suppression of interleukin-6 synthesis could, therefore, be of therapeutic value. Upon screening a number of substances, we found that a small number of nonsteroidal antiinflammatory drugs, including tenidap, were able to inhibit interleukin-6 synthesis in cultured human astrocytoma cells. These substances may be therapeutically useful in Alzheimer's disease and should be evaluated in clinical studies.

Heterogeneous turnover of terminal and core sugars within the carbohydrate chain of dipeptidylaminopeptidase IV isolated from rat liver plasma membrane.

Dipeptidylaminopeptidase IV, a plasma membrane-bound glycoprotein, is characterized by an intramolecular heterogeneous turnover of the protein backbone and carbohydrate chain. The faster turnover of the latter is restricted only to the outer sugars. The inner core sugars D-mannose and N-acetyl-D-glucosamine turn over at the same rate as the protein backbone.

Alpha 2-macroglobulin synthesis in interleukin-6-stimulated human neuronal (SH-SY5Y neuroblastoma) cells. Potential significance for the processing of Alzheimer beta-amyloid precursor protein.

Cultured human neuronal (SH-SY5Y neuroblastoma) cells synthesize and secrete the potent protease inhibitor alpha 2-macroglobulin (a2M) upon stimulation with interleukin-6 (IL-6) indicating that alpha 2-macroglobulin behaves as an acute-phase protein in the human central nervous system. Exogenous addition of a2M to the cultured neuronal cells resulted in only a slight inhibition of Alzheimer beta A4-amyloid precursor protein (APP) synthesis, but markedly inhibited its secretion pointing to the possibility that a2M may affect the proteolytic APP processing. Evidence is provided that IL-6 and a2M are involved in Alzheimer's disease pathogenesis.

In-vitro matured human macrophages express Alzheimer's beta A4-amyloid precursor protein indicating synthesis in microglial cells.

Microglia which are consistently associated with Alzheimer's disease (AD) senile plaques are part of the mononuclear phagocyte system. In-vitro matured human monocyte-derived macrophages feature many immunological characteristics of microglia. We found strong constitutive expression of Alzheimer's beta A4-amyloid precursor protein (APP) in human mononuclear phagocytes after terminal in-vitro maturation from monocytes to macrophages. Amyloid has previously been found to be associated with microglia in AD brains, however, it remained unclear whether the material was synthesized in or had been phagocytosed by the cells. The findings presented here support the assumption that brain microglia may contribute to APP synthesis in AD brain.

Expression of gamma delta T lymphocytes derived from human intestinal biopsies.

Although most T cells express the alpha/beta TCR, the gamma/delta TCR is expressed only on a small percentage of peripheral lymphocytes and CD3+ intestinal T cells. The most striking feature is a wide variation in the proportion of gamma/delta+ T cells in freshly isolated peripheral blood cells from normal individuals and patients with IBD. The augmentation of the gamma/delta+ T cell subpopulation derived from human intestinal biopsies after repeated stimulation with MT, even in the absence of filler cells, suggests that gamma/delta+ cells from human gut mucosa may play a role in generating a primary immune response to MT.

Studies of human myelin proteins during old age.

The electrophoretic protein patterns of myelin isolated from frontal and callosal white matter were studied in adult man up to the age of 90 years. The proportions of the four major myelin proteins remained virtually unchanged as did the total protein content of white matter and of purified myelin. The total mass of purified myelin that could be recovered from white matter gradually decreased with age, suggesting an age-related loss of myelin sheath and probably neurons as well, without detectable alterations of the regular protein composition of myelin. In most cases basic protein of myelin was preceded by one or two minor protein components on electrophoresis. One of them is tentatively identified as "prebasic" protein similar to the one previously observed in other species, because of its close electrophoretic apposition to the main basic protein. The second component was found less frequently and was thought to arise from specific types of proteolysis of myelin proteins. Prolonged time intervals between death and autopsy had little, if any, effect on the proportions of basic protein and proteolipid protein. Similar results were obtained when bovine brain was incubated under conditions designed to simulate post-mortem autolysis. It was there fore concluded that meaningful data on proteins of human central myelin may be obtained even though an autopsy was not performed within a few hours of death.

Polymorphous high-grade B cell lymphoma is the predominant type of spontaneous primary cerebral malignant lymphomas. Histological and immunomorphological evaluation of computed tomography-guided stereotactic brain biopsies.

In this retrospective study, a series of 54 patients (1982-1989) with sporadic primary cerebral malignant lymphomas is presented. All diagnoses were uniformly done on computed tomography-guided stereotactic brain biopsies according to histological criteria and immunomorphological data. In this series, the tumors were predominantly (25 of 48; 52%) classified as polymorphous high-grade blastic B cell lymphomas. This lymphoma type is therefore regarded as the most common type of sporadic primary cerebral non-Hodgkin's lymphoma. Severe regression (++/ ), which may dramatically alter the morphological appearance of a brain lymphoma, was found in 24 of 28 (86%) of cases with glucocorticoid administration prior to stereotactic brain biopsy.

Effects of interleukin-1 and interleukin-6 on metallothionein and amyloid precursor protein expression in human neuroblastoma cells. Evidence that interleukin-6 possibly acts via a receptor different from the 80-kDa interleukin-6 receptor.

Since immunohistochemical studies indicated the presence of interleukin-6 in the cortices of patients with Alzheimer's disease, we were interested in the eventual biological effects of this cytokine on neuronal cells. We found that interleukin-6 and interleukin-1 induced metallothionein expression in a human neuronal (SH-SY5Y neuroblastoma) cell line. In contrast to metallothionein, amyloid precursor protein expression was unaffected by both cytokines. When searching in the same cell line for the expression of the classical 80-kDa interleukin-6 binding protein, which is part of the dimeric interleukin-6 receptor, we were unable to detect the respective mRNA. Our findings either indicate that the interleukin-6 receptor in these cells is expressed in extremely low levels or that interleukin-6 may act upon neuronal cells via a different, yet unknown neuronal receptor.

Predominantly neuronal expression of cytochrome P450 isoforms CYP3A11 and CYP3A13 in mouse brain.

Despite the very small amounts of cytochrome P450 enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function, and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450 isoforms is determined by controversial interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the expression of cytochrome P450 3A11 and 3A13 in mouse brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed identical distribution of their proteins and mRNAs throughout the brain especially in neuronal populations, and to some extent in astrocytes. The cerebral expression of these P450 isoforms was confirmed by Western blot and RNAse protection assay analysis. The well-known testosterone-metabolizing capacity and the inducibility of cytochrome P450 3a isoforms by xenobiotics as well as their presence in steroid hormone-sensitive areas and neurons (e.g. hippocampus) clarify the significance of these isoforms for impairment of steroid hormone actions by P450-inducing environmental substances. Therefore, investigation of inducible cerebral P450 isoforms which are able to metabolize xenobiotics as well as steroid hormones might help us to understand neuroendocrine regulation of brain's plasticity.

Mapping of phenytoin-inducible cytochrome P450 immunoreactivity in the mouse central nervous system.

The distribution of phenytoin-inducible cytochrome P450 in non-treated mouse brain and spinal cord was analysed immunohistochemically using polyclonal antibodies against phenytoin-induced mouse cerebral microsomal P450. This P450 protein was proved in Ouchterlony [Volk B. et al. (1988) Neurosci. Lett. 84, 219-224], Western blot, and immunohistochemical analyses to be reactive to the specific antibodies and an IgG fraction raised against phenobarbital-induced rat liver microsomal P450IIB1. The phenytoin-induced P450 is designated P450IIB1* because immunologically it is comparable with P450IIB1; however, it has not yet been analysed for other characteristics of this enzyme. Immunocytochemistry was performed on acetone-fixed serial cryosections of the whole brain using the avidin-biotin-peroxidase detection system. Negative controls included incubations with preimmune serum of the immunized animal instead of the primary antibody and preabsorption of the antibody with the corresponding immunogen. The pattern of immunoreactive sites indicates that P450IIB1* is not distributed evenly throughout the CNS. It was found to be restricted to only some cellular populations. The most striking aspect of immunostaining was a predominant reactivity in the evolutionary old brain parts. Neuropil and neuronal staining was found in the spinal cord (motor neurons of the ventral horn), medulla oblongata (hypoglossal nuclei, magnocellular part of the lateral reticular nuclei), pons (trigeminal, facial, cochlear and pontine nuclei), cerebellum (granule cells), midbrain (dorsal raphe nucleus) and limbic lobe (hippocampal pyramidal cells). Neuropil reactivity alone appeared in cerebellar nuclei, midbrain, thalamus, basal ganglia, neopallium and olfactory brain. Generally, pia mater/arachnoid, ependyma, choroid plexus, vascular system and some astrocytic populations were found to be strongly P450IIB1* immunoreactive. In comparison with astroglia, which is characterized by glial fibrillary acidic protein-positiveness, the astrocytes, which are also P450IIB1* reactive, occurred only in subpial and subependymal layers, and in large fiber tracts of the spinal cord and brainstem, where they were attached to the vascular system. Otherwise, the glial fibrillary acidic protein-positive astrocytes were not P450IIB1* immunoreactive in the cerebellar molecular layer (fibers of Bergmann glia), in remaining neuropils and in white matter areas.

The risk of interstitial radiotherapy of low-grade gliomas.

BACKGROUND AND PURPOSE: The risk of side effects of low activity (i.e. <20 mCi) Iodine-125I (125I) interstitial radiotherapy was analyzed in patients with low-grade gliomas. MATERIALS AND METHODS: Permanent (247 patients) or temporary 125I-implants (268 patients) were used with a median reference dose of 60 Gy and 100 Gy, respectively, which was calculated to the outer rim of the tumour. The mean dose rate for temporary implants was low (median, 10 cGy/h). Risk factors were obtained from the multivariate proportional-hazards model. RESULTS: Radiogenic complications occurred in 39/515 patients (28 patients with transient symptoms and 11 patients with progressive symptoms). The most important risk factor was the volume of the intratumoural 200 Gy isodose. Available experimental data have associated a high dose zone in this range with the size of the treatment induced radionecrosis. Rapid tumour shrinkage (decrease of the tumour volume > or =50%) within the first 6 months with subsequent centripetal movement of non-pathologic tissue into the high dose zone and a reimplantation were additional risk factors. Radiation injury after rapid tumour shrinkage could be better avoided with temporary implants. A 200 Gy isodose volume <4.5 ml corresponded to an estimated risk of radiogenic complications <3%. There was a steep increase of the risk beyond this limit. Translation of the 200 Gy isodose volume in terms of the treatment volume and the reference dose allows rational treatment planning. The estimated risk of a temporary implant with an applied reference dose of 60 Gy and a treatment volume <23 ml was <3%. CONCLUSIONS: The intratumoural necrotizing effect of a low activity 125I implant limits its application to small treatment volumes. Radiation injury outside the treatment volume can be better avoided with temporary implants in the case of rapid tumour shrinkage.

The gangliosidoses.

The gangliosidoses are hereditary diseases with a recessive mode of inheritance and are caused by a genetically induced enzymatic block, which results in the accumulation of gangliosides in various tissues of the body, mainly in the brain. Although Tay-Sachs disease, the most commonly occurring of the gangliosidoses, has been known for nearly 100 years, additional variants of ganglioside "storage" disorders have been discovered during the past 15 years. Considerable progress in the knowledge of these disorders has been made with the advent of electron microscopy and with the elaboration of new biochemical and enzyme-chemical techniques. At the present the gangliosidoses are not amenable to therapy. Therefore the foreseeable future the pragmatic approach involves identification of the high-risk pregnancy and antenatal diagnosis.

Testosterone metabolism in rat brain is differentially enhanced by phenytoin-inducible cytochrome P450 isoforms.

Many cytochrome P450 (P450) isoforms are selectively inducible by xenobiotics, e.g. pharmaceuticals like the anti-epileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones and are of great importance, especially in early brain development. In this study, the hydroxylation of testosterone by rat brain microsomes from control and phenytoin-induced animals was examined by use of high performance liquid chromatography (HPLC) provided with a photodiode array detector (PDA). In control rats, testosterone is converted by cytochrome(s) P450 to 6alpha-hydroxytestosterone (OHT) as the main metabolite and 6beta-OHT as well as androstenedione as minor metabolites. After phenytoin treatment, brain microsomes showed a strong increase of testosterone metabolism to 2alpha-, 6beta-, 16alpha-, 16beta-OHT and androstenedione, whereby 16alpha-OHT was the main degradation product. These metabolites indicated the action of isoforms of the P450 subfamilies CYP2B, CYP2C and CYP3A. Inhibition experiments with antibodies against CYP2B1/2 and with the CYP2B specific inhibitor orphenadrine indicated the occurrence of members of this subfamily which are known to catalyse the oxidation of testosterone to 16alpha-OHT, 16beta-OHT and androstenedione. Western blots revealed the phenytoin-inducible expression of CYP2B1 and the constitutive expression of CYP3A. The latter is involved in the 6beta-hydroxylation of testosterone which was found correspondingly in control microsomes. Distinct CYP2C isoforms involved in the hydroxylation of testosterone in phenytoin-induced microsomes are not yet identified. The highly increased testosterone metabolism by phenytoin-dependent induction of specific cytochrome P450 isoforms in adult rat brain illustrates the potential influence of exogenous substances on internal regulative and metabolic pathways in the brain.