Etoposide Model-Induced Changes in Antioxidant Studies Histological and Ultrastructural Evaluations of Hepatic, Renal, and Cardiac Tissues of Male Rats / Cambios Inducidos por el Modelo de Etopósido en Estudios de Antioxidantes, Evaluaciones Histológicas y Ultraestructurales de Tejidos Hepáticos, Renales y Cardíacos de Ratas Macho

SUMMARY: Etoposide is an effective antimitotic and antineoplastic agent used to treat various human malignancies. In the present study, Etoposide was injected intraperitoneally into the rats at 1 mg/kg/day for 52 days (52 doses). The control animals received physiological saline (0.5 ml) intraperitoneally daily for 52 doses. The body weight of etoposide-treated rats was significantly reduced compared to control rats. Lipid peroxidation demonstrated an insignificant rise in hepatic tissue, a non-significant decline in renal tissue, and a significant reduction in cardiac tissue. The levels of GSH in hepatic and renal tissue were found to be non-significantly increased but significantly increased in cardiac tissue compared to controls. GR activity was found to be considerably decreased in the treated group. G-S-T levels increased significantly in all treated group. Etoposide injections caused a non-significant change in the GPX level of hepatic tissue, whereas renal and cardiac tissues showed a significant increase. The activity of CAT in hepatic tissue was significantly increased, while CAT activity in renal tissue showed a non-significant decrease, whereas in cardiac tissue, significantly lower levels were observed than in control group. The level of CYTp450 in hepatic and cardiac tissues showed a significant increase; however, renal tissue showed non-significant depletion, whereas CYTb5 in hepatic, renal, and cardiac tissues was significantly lower than controls. The protein content in the hepatic tissue was not significantly increased, whereas the total protein in the renal and cardiac tissues was increased significantly. The research finding is indicative of detoxification activity in the etoposide model.

El etopósido es un agente antimitótico y antineoplásico eficaz que se utiliza para tratar diversas neoplasias malignas humanas. En el presente estudio, se inyectó etopósido por vía intraperitoneal a las ratas a razón de 1 mg/kg/día durante 52 días (52 dosis). Los animales control recibieron solución salina fisiológica (0,5 ml) por vía intraperitoneal diariamente por 52 dosis. El peso corporal de las ratas tratadas con etopósido se redujo significativamente en comparación con las ratas del grupo control. La peroxidación lipídica demostró un aumento insignificante del tejido hepático, una disminución no significativa del tejido renal y una reducción significativa del tejido cardíaco. Se encontró que los niveles de GSH en el tejido hepático y renal no aumentaron significativamente, pero sí aumentaron significativamente en el tejido cardíaco en comparación con los controles. Se encontró que la actividad de GR disminuyó considerablemente en el grupo tratado. Los niveles de G-S-T aumentaron significativamente en todos los grupos tratados. Las inyecciones de etopósido provocaron un cambio no significativo en el nivel de GPX del tejido hepático, mientras que los tejidos renal y cardíaco mostraron un aumento significativo. La actividad de CAT en el tejido hepático aumentó significativamente, mientras que la actividad de CAT en el tejido renal mostró una disminución no significativa, mientras que en el tejido cardíaco se observaron niveles significativamente más bajos que en el grupo de control. El nivel de CYTp450 en los tejidos hepático y cardíaco mostró un aumento significativo; sin embargo, el tejido renal mostró un agotamiento no significativo, mientras que CYTb5 en los tejidos hepático, renal y cardíaco fue significativamente menor que los controles. El contenido de proteínas en el tejido hepático no aumentó significativamente, mientras que la proteína total en los tejidos renal y cardíaco aumentó significativamente. El hallazgo de la investigación es indicativo de la actividad de desintoxicación en el modelo de etopósido.

Mechanism of electron transfers mediated by cytochromes c and b5 in mitochondria and endoplasmic reticulum: classical and murburn perspectives.

We explore the mechanism of electron transfers mediated by cytochrome c, a soluble protein involved in mitochondrial oxidative phosphorylation and cytochrome b5, a microsomal membrane protein acting as a redox aide in xenobiotic metabolism. We found minimal conservation in the sequence and surface amino acid residues of cytochrome c/b5 proteins among divergent species. Therefore, we question the evolutionary logic for electron transfer (ET) occurring through affinity binding via recognition of specific surface residues/topography. Also, analysis of putative protein-protein interactions in the crystal structures of these proteins and their redox partners did not point to any specific interaction logic. A comparison of the kinetic and thermodynamic constants of wildtype vs. mutants did not provide strong evidence to support the binding-based ET paradigm, but indicated support for diffusible reactive species (DRS)-mediated process. Topographically divergent cytochromes from one species have been substituted for reaction with proteins from other species, implying the involvement of non-specific interactions. We provide a viable alternative (murburn concept) to classical protein-protein binding-based long range ET mechanism. To account for the promiscuity of interactions and solvent-accessible hemes, we propose that the two proteins act as non- specific redox capacitors, mediating one-electron redox equilibriums involving DRS and unbound ions.Communicated by Ramaswamy H. Sarma.

[The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins]. / Stepen' pokrytiia aminokislotnoi posledovatel'nosti pri ispol'zovanii razlichnykh metodov analiza mass-spektrometricheskikh dannykh, poluchennykh na model'nykh belkakh.

The aim of this study was to evaluate sequence coverage of five model proteins (CYB5A, SMAD4, RAB27B, FECH, and CXXC1) by means of shotgun proteomic data analysis employing different methods of data treatment including database-dependent search engines (MASCOT and X!Tandem) and de novo sequencing software ((PEAKS, Novor, and PepNovo+). In order to achieve maximal results, multiprotease hydrolysis including enzymes trypsin, LYS-C, ASPN and GluC was performed in solution and using the FASP method. High resolution mass spectrometry was carried out with a Q EXACTIVE HF hybrid mass spectrometer in the positive ionization mode; parent ions with the highest intensity and a charge range from +2 to +6 were fragmented in the HCD mode. 27 experiments were carried out (hydrolysis with each of 5 enzymes in solution, 4 for the FASP protocol, three technical repeats). Using parameters limiting false identification of peptides, the search engines and de novo sequencing software gave similar results. The degree of sequence coverage was not at least 40%, and in the best cases it reached 80-90%. The use of de novo sequencing software resulted in identification of the Y12H amino acid substitution in one model protein (CYB5A).

Using Photoconvertible and Extractable Fluorescent Proteins to Study Autophagy in Plants.

Several methodologies have been employed to understand the kinetics of induced autophagic degradation in plants, but most of them are not capable of distinguishing the autophagic cargo proteins before and after induction of autophagy in cells. Here, we designed a mass photoconverter that allowed us to simultaneously monitor protein synthesis and degradation in tobacco BY-2 cells using a photoconvertible fluorescence marker protein, Kikume Green Red (KikGR). An example of a new protocol for the analysis of autophagy progression using a fusion protein of cytochrome b5 and KikGR under phosphate starvation is described. The other example described is the analysis of the proliferation of Golgi apparatus in tobacco BY-2 cells using the fusion protein of a prolyl 4-hydroxylase NtP4H1.1 and monomeric KikGR. A detailed protocol on key analysis, as well as tips and notes for experiments using KikGR proteins, are described.

Photo-initiated crosslinking extends mapping of the protein-protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5.

Protein-protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein-protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules. This study is devoted to structure-functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450. After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the "bottom up" approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We found that all the above-mentioned cytochrome b5 Met residues can form intermolecular crosslinks and thus participate in the interaction. In addition, our results indicate the existence of at least two P450:cytochrome b5 complexes which differ in the orientation of individual proteins. The results demonstrate the advantages of the photo-initiated crosslinking technique which is able to map the protein-protein interfaces not only in the solvent exposed regions, but also in the membrane-embedded segments (compared to a typical crosslinking approach which generally only identifies crosslinks in solvent exposed regions).

Highly sensitive protein detection by combination of atomic force microscopy fishing with charge generation and mass spectrometry analysis.

An approach combining atomic force microscopy (AFM) fishing and mass spectrometry (MS) analysis to detect proteins at ultra-low concentrations is proposed. Fishing out protein molecules onto a highly oriented pyrolytic graphite surface coated with polytetrafluoroethylene film was carried out with and without application of an external electric field. After that they were visualized by AFM and identified by MS. It was found that injection of solution leads to charge generation in the solution, and an electric potential within the measuring cell is induced. It was demonstrated that without an external electric field in the rapid injection input of diluted protein solution the fishing is efficient, as opposed to slow fluid input. The high sensitivity of this method was demonstrated by detection of human serum albumin and human cytochrome b5 in 10(-17) -10(-18) m water solutions. It was shown that an external negative voltage applied to highly oriented pyrolytic graphite hinders the protein fishing. The efficiency of fishing with an external positive voltage was similar to that obtained without applying any voltage.

Complementary surface-enhanced resonance Raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles.

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b(5), whereas functionalization with SiO(2) amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

Phenobarbital influence on neuromuscular block produced by rocuronium in rats.

PURPOSE: To evaluate in vitro and in vivo neuromuscular blockade produced by rocuronium in rats treated with Phenobarbital and to determine cytochrome P450 and cytochrome b5 concentrations in hepatic microsomes. METHODS: Thirty rats were included in the study and distributed into 6 groups of 5 animals each. Rats were treated for seven days with phenobarbital (20 mg/kg) and the following parameters were evaluated: 1) the amplitude of muscle response in the preparation of rats exposed to phenobarbital; 2) rocuronium effect on rat preparation exposed or not to phenobarbital; 3) concentrations of cytochrome P450 and cytochrome b5 in hepatic microsomes isolated from rats exposed or not to phenobarbital. The concentration and dose of rocuronium used in vitro and in vivo experiments were 4 microg/mL and 0,6 mg/kg, respectively. RESULTS: Phenobarbital in vitro and in vivo did not alter the amplitude of muscle response. The neuromuscular blockade in vitro produced by rocuronium was significantly different (p=0.019) between exposed (20%) and not exposed (60%) rats; the blockade in vivo was significantly greater (p=0.0081) in treated rats (93.4%). The enzymatic concentrations were significantly greater in rats exposed to phenobarbital. CONCLUSIONS: Phenobarbital alone did not compromise neuromuscular transmission. It produced enzymatic induction, and neuromuscular blockade in vivo produced by rocuronium was potentiated by phenobarbital.

Gender and gonadal status differences in zona reticularis expression in marmoset monkey adrenals: Cytochrome b5 localization with respect to cytochrome P450 17,20-lyase activity.

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive.

Evaluation of chemopreventive action of Ginsenoside Rp1.

We evaluated the chemopreventive properties of Ginsenoside Rp1 on 7,12-Dimethyl benz (a) anthracene (DMBA) skin papillomagenesis in Swiss albino mice. A significant reduction in values of tumor incidence, tumor burden, and cumulative number of papilloma was observed in mice treated orally with Ginsenoside Rp1 continuously at pre-, peri- and post-initiational stages of papillomagenesis as compared to the control group. Chemopreventive potential of Ginsenoside Rp1 was also observed on the skin metabolizing enzymes in Swiss albino mice. Ginsenoside Rp1 produced a significant elevation in the skin microsomal cytochrome p-450 and cytochrome b5, glutathione S-transferase (GST), reduced glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), DT-diaphorase, superoxide dismutase (SOD) and catalase levels in the group of mice treated with Ginsenoside Rp1 for seven consecutive days. However, there was significant decrease in lipid peroxidation (LPO) level in Ginsenoside Rp1 treated group.

Enzymatic studies of cisplatin induced oxidative stress in hepatic tissue of rats.

Cisplatin is an active cytotoxic agent that has proved to be successful in the treatment of various types of solid tumors. The drug-induced nephrotoxicity has been very well documented in clinical oncology. However, hepatotoxicity has been rarely characterized and paid attention to, and is the least studied. We have used rat as the model to evaluate the effect of cisplatin on liver antioxidant enzymes and to determine whether these modulations in enzymatic activities are involved in hepatotoxicity. Reports obtained from our study indicate that cisplatin increases lipid peroxidation in the treated tissue of rat. The drug is also involved in altering the thiol status of the tissue with concomitant alterations in the enzymatic antioxidants. Glutathione and glutathione reductase levels were significantly decreased after cisplatin therapy, whereas glutathione peroxidase, gamma-glutamyl transpeptidase and catalase showed a significant increase. No statistically significant change was observed in glutathione-S-transferase activity. After cisplatin treatment, cytochrome P 450 showed a significant increase, whereas cytochrome b5 was decreased. Thus, an alteration in enzymatic antioxidant status with increase in lipid peroxidation indicates that the enzymes play an important role in combating free radical induced oxidative stress on the tissue.

Competitive ion-exchange adsorption of proteins: competitive isotherms with controlled competitor concentration.

The competitive adsorption processes inevitably present in chromatographic separations of complex mixtures have not been extensively studied. This is partly due to the difficulty of measuring true competitive isotherms, in which all system parameters (including competitor concentrations) are held constant. We report a novel approach to determining competitive protein adsorption isotherms in which the competitor concentration is held constant across the entire isotherm. By using the heme prosthetic group in cytochrome b5 as a quantitative spectrophotometric label, competitive isotherms between cytochrome b5 and alpha-lactalbumin can be constructed. Similarly, manganese-substituted protoporphyrin IX heme replacement allows the non-perturbing labeling of individual cytochrome b5 conservative surface charge mutants by replacement of a single atom in the interior of the protein. This labeling allows the study of competition between cytochrome b5 charge mutants of identical size and shape, which differ only in charge arrangement. Using these techniques, the effect of competing species on equilibrium behavior and the apparent heterogeneity of anion-exchange adsorbents in the presence of competitors can be quantitatively studied by fitting the data to two popular single-component binding models, the Temkin and the Langmuir-Freundlich (L-F) isotherms.

Effects of aging on cytochrome b5 expression in the human adrenal gland.

CONTEXT: Aging in humans is characterized by a selective decline in circulating levels of adrenal androgens. The results of in vivo studies are suggestive of reduced adrenal 17,20-lyase activity in aging men and women. OBJECTIVE: We sought to determine whether there are changes in the distribution and/or expression of cytochrome B5 (CytB5), an accessory protein important in the regulation of 17,20-lyase activity, in the adrenals of aging humans. DESIGN: Comparison between younger and older adrenal glands. SETTING: The study was conducted in a University Center. PATIENTS OR OTHER PARTICIPANTS: Adrenal glands obtained at autopsy after sudden death as a result of trauma from 46 young (age 20-40 yr) and 26 older (age 50-91 yr) humans were obtained and fixed within 24 h postmortem. INTERVENTIONS: Paraffin sections were stained with hematoxylin and eosin and also were subjected to immunohistochemical staining for CytB5. All sections were quantitatively evaluated using an image capture and analysis program and qualitatively evaluated with respect to staining intensity. MAIN OUTCOME MEASURES: To determine whether there are any changes in CytB5 distribution in the adult human adrenal cortex during the aging process using qualitative and quantitative analysis with respect to age, gender, race, and postmortem interval. RESULTS: CytB5 immunoreactivity was found in cells that corresponded to those of the zona reticularis. The percentage of the adrenal cortex immunoreactive for CytB5 decreased with aging (38.6 +/- 7.6% for young and 30.1 +/- 5.9% for older, mean +/- sd; P < 0.0001) as did the percentage of adrenocortical tissue comprising the zona reticularis (36.8 +/- 10.8% for young and 27.2 +/- 5.9% for older; P < 0.001). However, there was no apparent change in the staining intensity of CytB5 among those cells that were immunopositive for this factor with aging. CONCLUSIONS: There appears to be a reduction in the proportion of the adrenal cortex that expresses CytB5 with aging, and this likely corresponds to a shrinkage of the zona reticularis. The mechanism and cause for this cortical regression are unknown.

Unique supramolecular assembly of a redox protein with nucleic acids onto hybrid bilayer: towards a dynamic DNA chip.

Highly controlled supramolecular assemblies combining a genetically engineered redox protein, cytochrome b5, and modified oligonucleotides are presented. Modified b5 and DNA are covalently assembled through a hetero bifunctional cross-linker to give a unique hybrid molecular species. Moreover, the assembly includes a histidine tag head able to bind to modified phospholipids which lead to a new generation of self-assembled dynamic DNA chips. The interaction of the construction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using Biacore technology. The biochip, presented herein, features unique properties including tunable surface density of probes, very low non-specific interactions and optimization of hybridization efficiency. In addition, we demonstrated that the phase transition of the lipidic layer can modulate the dynamic of the association of the complex to the supported membrane. Potential applications of this new device are multiple including high sensitivity and high selectivity biochips, especially for studies of the DNA-ligands interactions in a biomimetic environment.

Dynamics of cytochrome P450 inducers in polluted sites of São Paulo city reservoirs.

The first analysis of water pollutants using biomarkers at the Guarapiranga Reservoir, which supplies water for one-third of the population of the São Paulo megalopolis (Brazil), is reported. Studies were performed before and after the start of water pumping to the Guarapiranga from the highly polluted Billings reservoir. Billings's water was purified by passing through the natural wetland located near Guarapiranga. Liver enzymes of Oreochromis niloticus (tilopias) obtained from both reservoirs served as biomarkers of pollution in a comparison with animals obtained from a reference site. Enhanced levels of total cytochromes P450 (3.4 times) and b5 (2.7 times) and activity of cytochrome c (P450) reductase (2.2 times) were observed in specimens collected near the water influx from the Billings before the pumping started. However, these parameters were significantly decreased 3 months later. This effect is probably due to dilution of pollutants because of the increased level of water in the Guarapiranga.

Effects of chronic exposure to cadmium on renal cytochrome P450-dependent monooxygenase system in rats.

The aim of this study was to evaluate the effects of chronic exposure to cadmium (Cd) on the renal cytochrome P450-dependent monooxygenase system. For this purpose, male Wistar rats were intoxicated with Cd administered in drinking water at a concentration of 5 or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b(5) as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b(5) reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b(5) (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b(5) and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b(5) and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40+/-0.15 microg/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.

Induction of P450 monooxygenases in the German cockroach, Blattella germanica L.

The cytochrome P450 monooxygenases are an important metabolic system whose level of activity can be influenced by several dietary constituents. We examined the effects of six known P450 inducers on the levels of total cytochromes P450, cytochrome b(5), and six monooxygenase activities in adult German cockroaches. In addition, the levels of CYP6L1 and CYP9E2 mRNA were also investigated. Phenobarbital treatment resulted in increases in total cytochromes P450 and metabolism of three resorufin analogues, but not CYP6L1 nor CYP9E2 mRNA. There was no significant effect of the other five inducers on any of the monooxygenase parameters we measured. In comparison with other insects, the German cockroach seems unusually refractory to most inducing agents.

Dual subcellular distribution of cytochrome b5 in plant, cauliflower, cells.

Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

Dilinoleoylphosphatidylcholine decreases ethanol-induced cytochrome P4502E1.

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b(5), total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.