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.

[Effects of hip arthroplasty followed by by inpatient rehabilitation on physical function and quality of life]. / Effekte der Hüftarthroplastik und der nachfolgenden stationären postoperativen Rehabilitation auf körperliche Funktion und Lebensqualität.

The aim of the present study was to investigate the effects of hip arthroplasty followed by an inpatient rehabilitation. Moreover, the relationships among functional status, quality of life and satisfaction with life or health status were examined. Patients were assessed before hip arthroplasty, at the start and at the end of the inpatient rehabilitation. Functional status was measured by using the WOMAC questionnaire and quality of life with the Medical Outcome Survey Short Form 36 (SF-36). Satisfaction was determined with a specific questionnaire (FLZ). Hip arthroplasty followed by an inpatient rehabilitation resulted in significant and clinically highly important improvements of functional status, quality of life and satisfaction with health and life.

Oxidative hydrolysis of scoparone by cytochrome p450 CYP2C29 reveals a novel metabolite.

Regioselective 7-demethylation of scoparone is regularly employed as an indicator of phenobarbital-like induction of rat liver cytochrome P450 isoform CYP2B1, e.g., by the antiepileptic drug phenytoin. After induction with phenobarbital and phenytoin, a new reaction sequence catalyzed by Cyp2c29 was identified in mouse liver microsomes. Cyp2c29-dependent 6-demethylation of scoparone resulted in the formation of isoscopoletin, an intermediate which is susceptible to further oxidation. This subsequent oxidation was also catalyzed by Cyp2c29 with a K(m) of 30,31 microM and a V(max) of 3,41 microM/min x microM P450, and resulted in the formation of the new metabolite 3-[4-methoxy-p-(3,6)-benzoquinone]-2-propenoate. This novel metabolite is the product of two consecutive oxidation reactions, proceeding over isoscopoletin to a putative lactone which is accessible to immediate hydrolysis, due to the onium character of the ring oxygen. This opening of the lactone ring corresponds to an oxidative hydrolysis. Differential oxidation of scoparone can be used as a sensitive indicator for distinguishing between different cytochrome P450 isoforms.

Possible function of astrocyte cytochrome P450 in control of xenobiotic phenytoin in the brain: in vitro studies on murine astrocyte primary cultures.

[4-(14)C]Phenytoin underwent a rapid cellular uptake by diffusion within 5 min when applied in a concentration of 10 microM to mouse brain astrocyte cultures. Subsequently, a slow linear increase of intracellular radioactivity indicated metabolic trapping of the drug, with final concentrations reaching 144 pmol phenytoin/mg protein in the astrocytes. Phenytoin levels from 1 to 10 microM decreased cell viability by 15%. The action of cytochrome P450 present in astrocytes in concentrations of 16-17 pmol P450/mg protein could explain these slight cytotoxic effects by generating intermediate metabolites of phenytoin. In contrast, concentrations of 50 microM strongly inhibited cell proliferation. A Cyp2c29 immunorelated P450 isoform was expressed in nearly all astrocytes in culture. Intracellular [4-(14)C]phenytoin was degraded to its major metabolites dihydrodiol, p-HPPH, and m-HPPH through a P450-dependent reaction with a specific activity of 0.66 pmol/min x mg protein, or 0.12 pmol/min x mg protein as measured in cell homogenates. These data underscore the importance of astrocytes as brain cells active in the detoxification of foreign substrates, but also in their toxification due to reactive metabolites generated during these metabolic processes. After diffusionary influx of drugs and other xenobiotics, the astrocyte P450 monooxygenases perform an essential role in the mediation of toxicity most frequently encountered in highly vulnerable neurons.

Effects of trap design and CO2 presentation on the measurement of adult mosquito abundance using Centers for Disease Control-style miniature light traps.

Centers for Disease Control miniature light traps augmented with CO2 provide an effective method of monitoring Culex abundance and may provide a useful supplement to New Jersey light traps used by the California Mosquito Surveillance Program. To assist in standardizing sampling protocols, the present research compared the catch of adult mosquitoes collected using 4 trap designs and 3 CO2 presentation methods. When augmented with dry ice, the Arbovirus Field Station (AFS) trap (consisting of a 3-in. fan mounted into a white polyvinyl chloride pipe and operated without a light source or rain shield) collected as many or more Culex females than similar traps purchased from John W. Hock and American Biophysics, or a trap with a 4.25-in. 2-bladed fan constructed by the Orange County Vector Control District (similar to the Encephalitis Virus Surveillance model distributed by Bioquip). Few blooded or gravid females and males were collected, indicating that CO2 released from the dry ice and not light probably was the primary attractant. Catch of Culex tarsalis females in traps baited with CO2 released at 0.5-1.5 liters/min from gas cylinders was significantly greater than in traps baited with dry ice, even though the CO2 release rates from the dry ice at dusk probably were comparable to that released from the cylinders and averaged 0.4-0).5 liters/min for the night. Traps baited with 0.5 liters/ min of CO2 gas released in 15 3- or 2-sec bursts per hour collected the fewest mosquitoes. In all experiments, trap location effects were significant and accounted for as much variability in catch size as trap design or CO2 presentation. Sampling efficiency of all trap designs or CO2 presentations were consistent over time, space, and different levels of mosquito abundance.

Comparative effectiveness of three adult mosquito sampling methods in habitats representative of four different biomes of California.

The effectiveness of New Jersey (NJ) light, dry ice baited, and gravid female traps for collecting adult mosquitoes was compared at representative habitats in the Coachella, San Joaquin, and Sacramento valleys and the Los Angeles basin of California. The NJ light traps effectively sampled Anopheles freeborni, Culex tarsalis, Psorophora columbiae, and several Aedes when abundance was high in rural areas with minimal competitive illumination. Dry ice-baited encephalitis virus surveillance or CDC style traps collected significantly more females of most species at most localities than did NJ light traps, regardless of background illumination. The Cummings modification of the Reiter gravid female trap baited with a bulrush (Schoenoplectus) infusion was the best method for collecting Culex pipiens complex females in most habitats. In the Los Angeles basin, gravid traps baited with bulrush infusion collected, on average, 4.5 times more Culex quinquefasciatus females than did traps baited with the Reiter infusion. The bulrush infusion in combination with the Cummings trap design seemed to provide resting site cues and collected males as well as empty and bloodfed females. Mosquito surveillance programs in California should include the systematic operation of dry ice-baited and gravid female traps to improve surveillance sensitivity for selected species in appropriate habitats.

Effect of phenytoin on cytochrome P450 2B mRNA expression in primary rat astrocyte cultures.

Studies on cytochrome P450 2B (CYP2B) in the brain have essentially been focused on protein characterization and regional distribution. Due to the high sequence homology between the closely related CYP2B1 and 2B2 isoforms and the low amounts of the corresponding mRNAs few efforts have been made to analyze the expression, regulation, and inducibility of these P450 genes in a specific cell type. In the present study, we investigated CYP2B mRNA expression in primary rat astrocyte cultures under the influence of the anti-epileptic drug phenytoin, which is known to be a CYP2B inducing agent in liver. In situ hybridization with a digoxigenin (DIG)-labeled cRNA probe demonstrated that 30-40% of the astrocytes strongly expressed a CYP2B mRNA-specific signal within the first week of cultivation. With increasing age (> 14 days) a greater percentage of cells (>90%) expressed mRNA for P450 2B. However, the level of transcriptional activity was substantially lower than in younger cultures. To discriminate between the 2B1 and 2B2 isoforms the reverse transcription/polymerase chain reaction (RT/PCR) procedures were proved for rat hepatic mRNA as a control assay. Subsequently, the application of this method on cultured astrocytes confirmed that these brain cells may express CYP2B1 mRNA. CYP2B2 mRNA could not be detected in astrocyte cultures at any age examined. Phenytoin led to the down regulation of CYP2B1 mRNA, which contrasts with the drug inducing effect on hepatic CYP2B1 and 2B2 levels. After 4 hr of exposure of phenytoin to the astrocytes no amplification product could be detected at all. Phenytoin did not induce either CYP2B1 or 2B2 expression.

Phenytoin toxicity in an older patient with slow metabolism and atypical presentation.

Phenytoin toxicity occurred in an older man who received a dosage of 300 mg/day. The patient developed high serum phenytoin concentrations from this common adult dosage, with symptoms of functional decline and altered mental status. These behaviors were perceived as sun-downing and were treated with haloperidol. A long hospitalization was required.

Time of host-seeking by Culex tarsalis (Diptera:Culicidae) in California.

Factors altering the pattern of Culex tarsalis Coquillett host-seeking activity were studied in Kern and Riverside counties of California using an automatic time-segregated sampler baited with bottled CO2 gas released at 0.5 or 1.0 liters/min. Host-seeking always commenced shortly after sunset and usually peaked during the succeeding 1-3 h, the hottest and driest time of the night. The time of maximal activity varied over time and space, because of increased mosquito abundance (presumably reduced blood feeding success), distance of the sampler from resting sites, adulticide applications, and perhaps weather.

Possible role of cytochrome P450 in inactivation of testosterone in immortalized hippocampal neurons.

The hippocampus as part of the limbic system is sensitive to gonadal hormones. The time-dependent expression of steroid receptors and the testosterone converting enzyme aromatase (CYP19) is well studied. In contrast, little is known about other cytochrome P450 enzymes in hippocampus which inactivate the gonadal hormones. For investigation of the total cytochrome P450 content and the expression of testosterone degrading CYP2B10 we used embryonic (E18) in comparison to postnatal (P21) immortalized hippocampal neurons. These embryonic neurons were demonstrated to react to hormones according a 'critical period' of sexual differentiation: testosterone treatment (1 microM to 5 microM in the culture medium) resulted in a decrease of beta-tubulin, as showed by immunocytochemistry and Western blotting. Measurements with reduced CO-difference spectrum elucidated that the P450 concentration in the embryonic neurons (10.2 pmol/mg protein; S.D. +/- 1.9) was twice as high as in the postnatal ones (5.2 pmol/mg protein; S.D. +/- 1.0). Correspondingly, a high value of the mitochondrial subfraction of approx. 141 pmol P450/mg protein was found in the embryonic neurons relative to the mitochondrial value of 37.7 pmol P450/mg protein in the postnatal neurons. Our results suggest a differential expression of cytochrome P450 during development. CYP2B10 was proved by electron microscopy and hormone degrading activity.

[Prévot nailing, an elegant method. Experiences with 38 personal cases]. / Die Prévot-Nagelung, eine elegante Methode. Erfahrungen mit 38 eigenen Fällen.

Endomedullar elastic stabilization with titanium Prévot nails is an excellent method of treating long-bone fractures in children. Without touching the epiphyseal line, this method guarantees early functional follow-up treatment with minimal operative trauma. For this reason, this method can compete successfully with common methods like extension treatment, external fixation and osteosynthesis with plates. From October 1990 to October 1993 we treated more than 40 fractures with Prévot nails and checked 38 of them clinically and radiologically. All 17 femur fractures healed correctly from an anatomical point of view and without rotation defects. In 4 patients we found a maximum difference in length of 1 cm in favor of the operated side. All of the 11 forearm fracture had good results, but there were 2 poor results with delayed union (adult patients) in the 6 humerus fractures operated upon. The remaining 4 cases, had to do with exceptional indications like lower leg fractures, combined humerus and forearm diaphyseal fractures, and metacarpal diaphyseal fractures. Our first experience with this method is good. The duration of hospitalization is short and early functional follow-up treatment for pediatric diaphyseal fractures is in general practicable. This procedure for operative care of femur and forearm diaphyseal fractures has now become the standard therapy in our clinic.

Localization and characterization of cytochrome P450 in the brain. In vivo and in vitro investigations on phenytoin- and phenobarbital-inducible isoforms.

The antiepileptic drug phenytoin is known to be substrate as well as inducer of cytochrome P450 (P450) in the mammalian liver. We were able to show the expression of P450 species immunorelated to the main phenytoin-induced hepatic isoforms in mice (CYP2C29) and rats (CYP2B1,2) also in the central and peripheral nervous system and primary cultures of cell types from the brain. The 2B1,2 related protein showed only a weak constitutive expression in vivo and in vitro analyzed by immunocytochemistry, in situ hybridization, Northern blot and RT/polymerase chain reaction (PCR). Contrary, the CYP2C29 related form is inducible by phenytoin at about 1.5-fold starting from an already higher constitutive level. This protein is characterized by a remarkable tendency to dissociate from the endomembranes during tissue homogenization. The supernatant of microsomal pellet is able to metabolize phenytoin in a reconstitutive system.

Occurrence of Culex (Melanoconion) erraticus (Diptera: Culicidae) in California.

Culex erraticus is added to the 52 currently recognized mosquito species from California. Four females and one male were collected in a CO2 trap on August 29, 1994, at the Imperial Wildlife Refuge, Wister Unit, Imperial County, CA. Additional specimens were collected on October 24, and larval habitats were located on October 31, 1994. Possible routes of introduction are discussed.

Effect of temperature on the transmission of western equine encephalomyelitis and St. Louis encephalitis viruses by Culex tarsalis (Diptera: Culicidae).

The extrinsic incubation rate (inverse of the time in days from infection to median transmission) of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses by laboratory strains of Culex tarsalis Coquillett increased as a linear function of incubation temperatures from 10 to 30 degrees C. The estimated temperatures for zero transmission thresholds (intercept of the X axis) were 10.9 and 14.9 degrees C, and the number of degree days above these thresholds required for median transmission (inverse of the slope) was 67.6 and 115.2, respectively. Although the bodies of most Cx. tarsalis females remained infected and the WEE viral titer did not decrease significantly throughout the incubation periods at all temperatures, transmission rates by females incubated at 20 to 30 degrees C decreased markedly after peaking at 7-10 d after infection. In contrast, midgut escape and salivary gland infection barriers limited the transmission rates of SLE virus at all temperatures, but these rates did not decrease markedly as a function of incubation time, indicating that virus modulation did not occur. Degree-day models were used to calculate monthly changes in the duration of the extrinsic incubation period for WEE and SLE viruses in the San Joaquin and Coachella valleys based on mosquito temperatures estimated by combining nocturnal air and diurnal resting site temperatures. Temperatures in the San Joaquin Valley averaged 5 degrees C cooler than in the Coachella Valley, proportionately shortening the duration of the potential transmission season for WEE virus from 10 to 8 mo and for SLE virus from 8 to 5 mo, respectively.

Mosquito and arbovirus ecology in southeastern California, 1986-1990.

Mosquito abundance and western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) virus activity were monitored in five valleys in southeastern California from June 1986 through April 1990 to study virus overwintering and possible dissemination from south to north along geographically defined corridors. Culex tarsalis Coquillett predominated in CO2 trap collections and was the only species repeatedly infected with WEE and SLE viruses. Abundance peaked during April-May and August-October. WEE virus infections in Cx. tarsalis generally were detected after the spring peak and were followed approximately 1 mo later by seroconversions in sentinel chickens. SLE virus infections occurred later in the summer but before the fall peak in Cx. tarsalis abundance. Peak Cx. tarsalis abundance occurred when monthly temperatures averaged 25 degrees C, whereas virus infections generally were detected most frequently when temperatures exceeded 29 degrees C. Although the spring increase in Cx. tarsalis abundance occurred earlier in southern valleys, the onset of virus activity was variable among valleys and did not follow a south to north progression.

Ecological observations on the 1989 outbreak of St. Louis encephalitis virus in the southern San Joaquin Valley of California.

Temporal and spatial patterns of St. Louis encephalitis (SLE) virus transmission were compared at permanent study areas in the southern San Joaquin Valley during years with low (1988 and 1990) and elevated (1989) viral activity. During 1989 and 1990, virus appeared first at sentinel chicken flocks exhibiting low to moderate seroconversion rates at the end of the previous season. This finding, and the early season seroconversion of sentinel chickens at a marsh habitat on 5 March and 2 April 1990, circumstantially indicated that SLE virus may have overwintered on the valley during the winters of 1988-1989 and 1989-1990. The mechanism of overwintering was not elucidated further, because virus could not be isolated from overwintering adult mosquitoes or from immatures collected during the spring. An outbreak of 26 confirmed SLE cases occurred in 1989 during a drought year (rainfall 50% of normal) and followed a spring with elevated temperatures (1.7-3.4 degrees C above normal) and Culex tarsalis Coquillett abundance. Cx. tarsalis was the primary vector, being most abundant during the virus amplification period in early summer and most frequently infected (70 SLE virus positive pools/329 tested). SLE virus also was detected in Culex quinquefasciatus Say (14/65) and Cx. stigmatosoma Dyar (1/4); however, both species were distributed focally and increased in abundance only after widespread seroconversions had occurred in sentinel chickens. Increased virus activity during 1989 was not accompanied by marked changes in vector susceptibility or in SLE virus infectivity for mosquitoes. Decreased virus activity in the Bakersfield area during 1990 could not be attributed to immunity in passeriform birds, because a small seroprevalence survey indicated that few adult birds had antibodies to SLE virus.

Population dynamics of adult Culex mosquitoes (Diptera: Culicidae) along the Kern River, Kern County, California, in 1990.

The temporal and spatial abundance, dispersal, survivorship, and density of Culex tarsalis Coquillett and Cx. quinquefasciatus Say populations were studied in riparian, agricultural, and residential habitats along the Kern River bed during the drought year of 1990. The temporal abundance of both species was related to cotton agricultural practices and peaked during intensive irrigation after cultivation was terminated in July. Cx. tarsalis peaked in abundance 2 wk earlier than Cx. quinquefasciatus, perhaps because of the advantage of autogenous oviposition, which shortened generation time. Although host-seeking females of both species were most abundant in the riparian habitat, more Cx. quinquefasciatus than Cx. tarsalis were collected in the residential habitat. Marked females released within the riparian habitat were recaptured most frequently within 1 km of the release point; however, flights as far as 12.6 km were documented within the 180-km2 study area. On average, marked Cx. quinquefasciatus dispersed farther and more rapidly than Cx. tarsalis and were recaptured more frequently within the residential habitat. Survivorship, estimated horizontally from the female recapture rate, ranged from 0.60 in May to 0.79 in July for Cx. tarsalis and from 0.74 in September to 0.84 in July for Cx. quinquefasciatus. The estimation of survivorship vertically from the parity rate was complicated by elevated autogeny rates in Cx. tarsalis and by the low parity rate in Cx. quinquefasciatus. Cx. tarsalis population density ranged from 125 females per km2 in May to 65,500 per km2 in August, and was well correlated with relative abundance. Collectively, these ecological data indicated that Cx. tarsalis may be important in disseminating arboviruses within the riparian habitat, but that Cx. quinquefasciatus may be important secondarily by disseminating virus from the riparian habitat to the adjacent residential habitat.

Urbanization and the efficiency of carbon dioxide and gravid traps for sampling Culex quinquefasciatus.

The efficiency of gravid and CO2 traps for sampling female Culex quinquefasciatus was evaluated along 2 parallel 6.4 km long urban (high housing density) to rural (low housing density) transects in east Bakersfield, Kern County, CA. There were no significant differences in the number of female Cx. quinquefasciatus collected by gravid traps within urban and rural zones. The number of females collected per trap night ranged from 6.8 to 15.5. The number of females collected by CO2 traps increased significantly from 1.4 to 3.1 per trap night in urban to 31.8 to 111.2 per trap night in rural zones and was inversely correlated with housing density. These results indicate that female Cx. quinquefasciatus were effectively sampled by gravid traps in urban subdivisions and by CO2 traps in rural mixed agricultural areas.

Influence of vegetation on carbon dioxide trap effectiveness for sampling mosquitoes in the Sierra Nevada foothills of Kern County, California.

The effect of vegetation on sampling Culex tarsalis, Cx. quinquefasciatus and Aedes nigromaculis by CO2 traps was evaluated at an intermittent stream habitat at the base of the Sierra Nevada foothills. Carbon dioxide traps were spaced along a 450 m transect perpendicular to Poso Creek to determine female attraction to traps placed in 5 different vegetation substrates: 1) open hilltop with sparse growth of grasses and saltbush, 2) open pasture with sparse growth of saltbush, 3) peripheral understory of mule fat, 4) shaded understory of mule fat, and 5) open canopy 5 m above ground in willow and cottonwood trees. Most host-seeking Cx. tarsalis and Cx. quinquefasciatus females were collected within the open canopy and peripheral understory. Host-seeking Ae. nigromaculis females were collected predominately in the open pasture and within the peripheral understory. The association between CO2 trap catch size and vegetation suggested a relationship between the host-feeding patterns and associated hunting strategies of these bird and mammal feeding species.