Biomedical subjects

F Oesch

Publications and source records attributed to F Oesch.

At least 19 recordsLinked to original sources

PubMed2005

Phosphorylation of cytochromes P450: first discovery of a posttranslational modification of a drug-metabolizing enzyme.

Cytochromes P450 (CYP) are important components of xenobiotic-metabolizing monooxygenases (CYP-dependent monooxygenases). Their regulation by induction, most commonly by transcriptional activation, mediated by xenobiotics, normally substrates of the corresponding CYP, is well known and has been widely studied. Our team has discovered an additional important regulation of xenobiotic-metabolizing CYPs pertaining to posttranslational modification by phosphorylation. Individual CYPs are phosphorylated by different protein kinases, leading to CYP isoenzyme-selective changes in the metabolism of individual substrates and consequent drastic changes in the control of genotoxic metabolites. Best studied are the CYP phosphorylations by the cAMP-dependent protein kinase A. Most recently, we discovered that cAMP not only leads to drastic changes in the activity of individual CYPs, but also to drastic changes in the nuclear localization of the CYP-related transcription factor Ah receptor (AHR). The consequences are very different from those of AHR nuclear translocation mediated by the classical ligands (enzyme inducers such as dioxin) and are likely to represent the long-sought physiological function of the AHR, its persistent disturbance by long-lived ligands such as dioxin may well be the reason for its high toxicity.

Animals↗

PubMed2005

Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction.

An important application of hepatocyte cultures is identification of drugs acting as inducers of biotransformation enzymes that alter metabolic clearance of other therapeutic agents. In the present study we optimized an in vitro system with hepatocytes cultured in alginate microspheres that allow studies of enzyme induction with excellent sensitivity. Induction factors obtained with standard inducers, such as 3-methylcholanthrene or phenobarbital, were higher compared to those with conventional hepatocyte co-cultures on collagen coated dishes. This is illustrated by activities of 7-ethoxyresorufin-O-deethylase (EROD) after incubation with 5 microM 3-methylcholanthrene (3-MC), a standard inducer for cytochrome P4501A1 and 1A2. Mean activities for solvent controls and 3-MC exposed cells were 2.99 and 449 pmol/min/mg protein (induction factor: 150) for hepatocytes cultured in microspheres compared to 2.72 and 80.6 pmol/min/mg (induction factor: 29.6) for hepatocytes on collagen coated dishes. To compare these in vitro data to the in vivo situation male Sprague Dawley rats, the same strain that was used also for the in vitro studies, were exposed to 3-MC in vivo using a protocol that guarantees maximal induction. Activities were 29.2 and 1656 pmol/min/mg in liver homogenate of solvent and 3-MC treated animals (induction factor: 56.7). Thus, the absolute activities of 3-MC exposed hepatocytes in microspheres are lower compared to the in vivo situation. However, the induction factor in vitro was even higher compared to the in vivo situation (150-fold versus 56.7-fold). A similar scenario was observed using phenobarbital (0.75 mM) for induction of CYP2B and 3A isoenzymes: induction factors for testosterone hydroxylation in position 16beta were 127.5- and 50.4-fold for hepatocytes in microspheres and conventionally cultured hepatocytes, respectively. The new in vitro system with hepatocytes embedded in solid alginate microspheres offers several technical advantages: (i) the solid alginate microspheres can be liquefied within 60s, allowing a fast and complete harvest of hepatocytes; (ii) alginate capsules are stable allowing transport and mechanical stress; (iii) high numbers of hepatocytes can be encapsulated in short periods; (iv) defined cell numbers between 600 hepatocytes, the approximate number of cells in one capsule, and 18 x 10(6) hepatocytes, the number of hepatocytes in 6 ml alginate, can be transferred to a culture dish or flask. Thus, encapsulated hepatocytes allow a flexible organization of experiments with respect to cell number. In conclusion, we optimized a technique for encapsulation of hepatocytes in alginate microspheres that allows identification of enzyme induction with an improved sensitivity compared to existing systems.

Alginates↗

PubMed2004

Metabolism of propafenone and verapamil by cryopreserved human, rat, mouse and dog hepatocytes: comparison with metabolism in vivo.

In the present study we examined the metabolism of [(14)C]propafenone (P) and [(14)C]verapamil (V) using cryopreserved human, dog (Beagle), rat (Sprague-Dawley) and mouse (NMRI) hepatocytes. The percentage ratios of the metabolites were identified after extraction by HPLC with UV and radioactivity detection. Phase-II metabolites were cleaved using beta-glucuronidase. Metabolism of the drugs by cryopreserved hepatocytes was compared with that in the respective species in vivo. All phase-I and -II metabolites known from in vivo experiments: 5-hydroxy-P (5-OH-P); 4'-hydroxy-P (4'-OH-P); N-despropyl-P (NdesP) and the respective glucuronides, were identified after incubation with cryopreserved hepatocytes. Interspecies differences were observed concerning the preferential position of propafenone hydroxylation: 5-OH-P made up 91, 51, 16 and 3% of the total metabolites after incubation with cryopreserved human ( n=4), dog ( n=3), rat ( n=3) and mouse ( n=4) hepatocytes respectively. These results are consistent with interspecies differences known from in vivo experiments. The metabolism of V is more complex than that of P. Nevertheless, all phase-I metabolites known from in vivo experiments and the expected glucuronides were identified after incubation with cryopreserved hepatocytes from all four species. As expected from the results of in vivo experiments, there were no major interspecies differences with respect to phase-I metabolites although the conjugation of verapamil phase-I metabolites by cryopreserved canine hepatocytes was much weaker than for the other species. In conclusion, phase-I and phase-II metabolism of P and V was evaluated using hepatocytes in vitro. All of the relevant interspecies differences known from in vivo experiments were identified after short-term incubation with cryopreserved hepatocytes in suspension.

Aged↗

PubMed2004

RNAi knock-down mice: an emerging technology for post-genomic functional genetics.

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene function in the mouse. Reviewed topics will include delivery methods for RNAi-mediating molecules, a comparison between traditional knock-out and innovative transgenic RNAi technology and the generation of graded RNAi knock-down phenotypes. Apart from the exciting possibilities RNAi provides for studying gene function in mice, we discuss several caveats and limitations to be considered. Finally, we present prospective strategies as to how RNAi technology might be applied for generating conditional and tissue-restricted knock-down mice.

Alleles↗

PubMed2002

2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent release from contact inhibition in WB-F344 cells: involvement of cyclin A.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of TCDD actions are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In rat liver epithelial WB-F344 cells, TCDD induces a release from contact inhibition, which is manifested by a twofold increase in cell number when TCDD (1 nM for 48 h) is added to confluent cells in the presence of serum, but not when given to exponentially growing or subconfluent, serum-deprived WB-F344 cells. Loss of G1 arrest was also shown by flow cytometric analysis. We demonstrate that TCDD treatment significantly increases cyclin D2 and cyclin A protein levels and show by immunofluorescence that these proteins accumulate in the nucleus. Although TCDD treatment leads to a strong increase in cyclin D2/cdk4 and cyclin A/cdk2 complex formation, we could only detect an elevation of cyclin A/cdk2 activity. In accordance with a lack of elevated cdk4 activity, no decrease in the amount of hypophosphorylated retinoblastoma protein could be shown after TCDD treatment. The importance of increased cyclin A/cdk2 activity for TCDD-dependent release from contact inhibition was shown by the fact that the cdk2/cdc2-specific inhibitor olomoucine (25 microM) abolished TCDD response. These data indicate cyclin A-dependent loss of G1 arrest after TCDD treatment mainly downstream of the retinoblastoma protein.

Animals↗

PubMed2002

Downregulation of beta2-microglobulin in human cord blood somatic stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells?

Adherently growing, non-hematopoietic somatic stem cells isolated from human cord blood were stained with the fluorescent dye PKH26 and transplanted into livers of SCID-mice to examine a possible cell fate transition. Already 7 days after transplantation stem cells were well integrated into the liver tissue. Human albumin that was not expressed by the stem cells before transplantation was detectable in the host's livers after injection of cord blood stem cells. Human alpha1-antitrypsin was detectable in stem cells already before transplantation and remained positive in the mouse liver. The most interesting observation in this study was the downregulation of human beta2-microglobulin (beta2M) in the stem cells after transplantation: beta2M is expressed constitutively in our cord blood stem cells. However, beta2M was no longer detectable by RT-PCR in all tissues where human albumin and alpha1-antitrypsin were expressed after stem cell transplantation. beta2M is known to participate as an integral part of the major histocompatibility complex. Absence of beta2M makes the residual heavy chain inactive as an antigen. Thus, downregulation of beta2M may represent an escape mechanism from killer-T cells and may be a molecular mechanism explaining the recently described "immunological blindness" [37] of stem cells. In contrast to the results obtained after direct injection of stem cells as a suspension, no consistent downregulation of beta2M was observed after transplantation of stem cells encapsulated in alginate beads to generate a compartment where stem cells are protected from the host's natural killer cells. No expression of human genes was observed after transplantation of human cord blood derived mononuclear cells (MNC) that were used as a negative control. In conclusion, we have shown that human cord blood somatic stem cells survive and are reprogrammed after transplantation into mouse livers, although a complete transdifferentiation to hepatocytes did not occur within 7 days, since some marker genes (GATA4 and alpha-fetoprotein) were still negative. Switching off expression of beta2M may be part of an intriguing and novel mechanism explaining why stem cells escape the host's immune system.

Aged↗

PubMed2002

Challenging dogma: thresholds for genotoxic carcinogens? The case of vinyl acetate.

Although many questions remain unanswered, the general principle of the sequence of events leading to cancer after exposure to genotoxic carcinogens has become increasingly clear. This helps to understand the parameters that influence the shape of the dose-effect curve for carcinogenesis, including metabolic activation and inactivation of carcinogens, DNA repair, cell cycle control, apoptosis, and control by the immune system. A linear dose-response relationship with no observable threshold seems to be a conservative but adequate description for the carcinogenic activity of many genotoxic carcinogens, such as aflatoxin B1, the tobacco-specific nitrosoketone NNK, and probably N,N-diethylnitrosamine. However, extrapolation models connecting the high-level risk to the zero intercept have clearly resulted in overestimations of risk. Vinyl acetate is an example that is discussed extensively in this review. At extremely high and toxic doses, vinyl acetate is carcinogenic in rats and mice and causes chromosomal aberrations. In tissues of contact, vinyl acetate is converted to acetic acid and acetaldehyde. Only when threshold levels are achieved do critical steps in the mechanism ultimately leading to cancer become active, namely pH reduction in exposed cells of more than 0.15 units leading to cytotoxicity, damage to DNA, and regenerative proliferation. Consistent with the known exposure to endogenous acetic acid and acetaldehyde, tissues sustain a certain level of exposure without adverse effects. Physiological modeling shows that the conditions necessary for carcinogenesis are in place only when threshold levels of vinyl acetate are exceeded. The example of vinyl acetate underlines the importance of toxicological research that unequivocally identifies genotoxic carcinogens acting through a threshold process.

Animals↗

PubMed2002

Permissive and suppressive effects of dexamethasone on enzyme induction in hepatocyte co-cultures.

1. Steroids are known to act as permissive factors in hepatocytes. This study shows that dexamethasone (DEX) is a permissive factor for induction of CYP2B1/2, CYP3A1, CYP2A1 and probably also CYP2C11 in cultures with primary rat hepatocytes. 2. The induction factor of phenobarbital (PB)-induced formation of 16beta-hydroxytestosterone (OHT), a testosterone biotransformation product predominantly formed by CYP2B1, is increased 18-fold by the addition of 32 nM DEX to the culture medium. Interestingly, higher concentrations of DEX up to 1000 nM led to a concentration-dependent maximally 5-fold decrease (p = 0.002) of phenobarbital-induced 16beta-OHT formation compared with the effect observed with 32 nM DEX. Thus, DEX shows permissive and suppressive effects on enzyme induction depending on the concentration of the glucocorticoid. 3. Qualitatively similar but smaller permissive and suppressive effects of DEX were observed for PB-induced CYP3A1 activity as evidenced by formation of 2beta-, 6beta- and 15beta-OHT. 4. DEX is a permissive factor for induction of CYP2A1 activity by 3-methylcholanthrene (3MC), as evidenced by the formation of 7alpha-OHT. Without addition of DEX, 3MC did not induce formation of 7alpha-OHT, whereas an almost 3-fold induction occurred in the presence of DEX. In contrast to CYP2B and CYP3A, concentrations up to 1000 nM DEX were not suppressive for the induction of CYP2A1. 5. We described recently a technique that allows preparation of cultures from cryopreserved hepatocytes. An almost identical influence of dexamethasone on enzyme induction was observed here in cultures from cryopreserved compared with freshly isolated hepatocytes. 6. Cultures with primary hepatocyte cultures represent a well-established technique for the study of drug-drug interactions. However, a large interlaboratory variation is known. Our study provides evidence that differences in glucocorticoid concentration in the culture medium contribute to this variation.

Animals↗

PubMed2001

cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal control in vitro and in vivo.

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status.

Animals↗

PubMed2001

Involvement of protein kinase Cdelta in contact-dependent inhibition of growth in human and murine fibroblasts.

There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCdelta-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCdelta in contact-inhibition. In accordance, the PKCdelta specific inhibitor Rottlerin (1 microM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCdelta to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCdelta in response to cell-cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCdelta-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCdelta induced a transformed phenotype. We conclude that PKCdelta is involved in contact-dependent inhibition of growth.

3T3 Cells↗

PubMed2001

DNA repair activity of 8-oxoguanine DNA glycosylase 1 (OGG1) in human lymphocytes is not dependent on genetic polymorphism Ser326/Cys326.

8-oxoguanine DNA glycosylase 1 (OGG1) is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8oxoG) from DNA. Since 8oxoG is a highly mispairing lesion, decreased OGG1 expression level could lead to a higher background mutation frequency and could possibly increase the cancer risk of an individual under oxidative stress. In order to analyse the natural variation of OGG1, we measured the DNA repair activity in human lymphocytes of healthy individuals by means of an 8oxoG-containing oligonucleotide assay. The data obtained revealed a two fold interindividual variation of OGG1 activity in lymphocytes. There was no difference in OGG1 activity due to gender and smoking behaviour. Transcriptional analyses of OGG1 showed the expression of two isoforms, 1a and b, in lymphocytes. Structural analysis of the human OGG1 (hOGG1) gene revealed a Ser326/Cys326 polymorphism in the Caucasian population with allele frequencies of 75% for Ser326 and 25% for Cys326. This polymorphism was not associated with altered OGG1 activity. The described routine test system for measuring OGG1 activity in cryopreserved lymphocytes provided highly reproducible results and is a useful tool for risk assessment associated with alterations in the repair of oxidative DNA damage.

Alleles↗

PubMed2001

c-erbB-2 expression in small-cell lung cancer is associated with poor prognosis.

Small-cell lung cancer (SCLC) carries a bad prognosis despite good initial response to chemotherapy. It is therefore important to identify molecular markers that influence survival as potential new therapeutic targets. In our study, expression of the tyrosine kinase c-erbB-2 (HER2/neu) receptor in tumor tissues of 107 consecutive newly diagnosed patients with primary SCLC was quantified using a monoclonal antibody directed against the c-terminal domain of c-erbB-2. A clear-cut positive expression of c-erbB-2 was observed in 13% of patients. Surprisingly, c-erbB-2 was an independent prognostic factor (RR = 2.16; p = 0.014) when a proportional-hazard model was adjusted to stage (limited vs. extensive disease) and performance status (WHO I-IV), the most relevant clinical parameters. Similarly, a significant association between c-erbB-2 and survival was obtained if a larger number of clinical parameters were included into the analysis, namely response to chemotherapy, TNM stage, lactate dehydrogenase (LDH), neuron-specific enolase (NSE), gender and age (p = 0.033). Interestingly, c-erbB-2 expression was more relevant for patients with advanced tumors. In the subgroup of patients with bad performance status (WHO II-IV), median survival of patients with undetectable c-erbB-2 expression was 274 days compared with only 23 days for patients with clear-cut positive c-erbB-2 immunohistochemistry (p = 0.0031; log-rank test). Similar results were obtained for patients with extensive disease (p = 0.028) and high TNM stages (T>2 or N>1 or M1; p < 0.068, all comparisons). In contrast, c-erbB-2 expression was not associated with survival in patients with limited disease (p = 0.97), low TNM stages (p > 0.56, all comparisons) and good performance status (p = 0.97). In conclusion, c-erbB-2 is expressed in more than 10% of SCLC. Expression of c-erbB-2 is an independent prognostic factor of survival. The effect of c-erbB-2 expression seems to become more important in advanced stages of the disease. Since c-erbB-2 is a therapeutical target in other types of cancer, further studies to identify the role of c-erbB-2 in SCLC are clearly warranted.

Age Factors↗

PubMed2001

Rottlerin induces a transformed phenotype in human keratinocytes.

PKCdelta plays a fundamental role in cell cycle control. Consistent with its proposed tumour suppressor function, ras transfection of the human keratinocyte cell line HaCaT results in a loss of PKCdelta expression mediated by TGFalpha (Exp. Cell Res., 219, 299, 1995). To get more insight into the role of PKCdelta in keratinocytes, we investigated the effects of Rottlerin, a specific inhibitor of protein kinase Cdelta, in HaCaT cells. After Rottlerin treatment, HaCaT cells lost their cobble-stone morphology and displayed a spindle-shaped, fibroblastic phenotype. Additionally, the establishment of cell-cell contacts was prevented. This was caused by an internalization of E-cadherin and beta-catenin as assessed by immunofluorescence. A similar phenotype was observed in the presence of a neutralizing anti-E-cadherin antibody. Rottlerin-treated HaCaT cells proliferated like transformed cells in a three-dimensional cell culture system. We therefore conclude that PKCdelta is involved in mediating cell-cell contacts via E-cadherin and hence regulates differentiation in HaCaT cells.

Acetophenones↗

PubMed2001

Metallothionein expression in ovarian cancer in relation to histopathological parameters and molecular markers of prognosis.

Metallothioneins (MTs) and glutathione constitute the major fractions of intracellular thiol factors. Abundant nucleophilic sulfhydryl groups can interact with many electrophilic substances, including several anti-neoplastic agents, participate in controlling intracellular redox potential, and act as scavengers of reactive oxygen species. In the present study, we examined the relation of MTs (alone and in combination with glutathione) to histopathological parameters and survival time of ovarian cancer patients. Expression of the major MT isoforms (MT-1 and MT-2) was determined by immunohistochemistry on paraffin-embedded tumor specimens from 189 patients, 151 suffering from primary epithelial ovarian cancer and 38 from recurrences. MT was negatively associated with survival time when all patients with primary carcinomas (n = 151) were analyzed (p = 0.049, log-rank test). However, no significant association between MT expression and survival was obtained when subgroups of patients with histological grade 1, 2 or 3 carcinomas were analyzed. Similarly, no significant association of MT expression and survival was obtained with the proportional hazards model adjusted for histological grade. This scenario can be explained by a correlation between MT expression and histological grade: MT was detectable in 26%, 48% and 62% of grade 1, 2 and 3 carcinomas, respectively (p = 0.008, chi(2) test). An interesting hypothesis is generated by combined analysis of MT and total glutathione content (GSH). The product of MT and GSH levels (MT x GSH) was negatively associated with survival of grade 1 carcinomas (p = 0.021, log-rank test) but not with grade 2 and 3 carcinomas (p = 0.176 and 0.403, respectively). When MT x GSH was greater than the median, 25% of patients with grade 1 carcinomas died within 235 days. In contrast, all patients with grade 1 carcinomas survived when MT x GSH in tumor tissue was smaller than the median. This suggests that high expression of sulfhydryl factors might facilitate survival and progression of low-grade ovarian cancer cells. A significant correlation was obtained between MT expression and mutant p53 (p = 0.037, chi(2) test). However, this might be an indirect effect since both MT (p = 0.008) and mutant p53 (p = 0.000) were associated with histological grade. In conclusion, MT expression as well as the product of MT and GSH were associated with histological grade of primary ovarian carcinomas. High expression of both sulfhydryl factors may identify a subgroup of low-grade carcinomas with an increased risk of progression.

Disease Progression↗

PubMed2001

Lack of mutagenic and co-mutagenic effects of magnetic fields during magnetic resonance imaging.

Mutagenic and co-mutagenic effects of static, pulsed bipolar gradient, and high-frequency magnetic fields, as well as combinations of them, were examined using the Ames test. The Ames test using Salmonella typhimurium bacteria, wild-type strain RTA, preincubation assay, without metabolic activation, was performed. All combinations of magnetic fields were tested with and without co-exposure to N-methyl-N'-nitro-N-nitrosoguanidine and benzo[a]pyrene-4,5-oxide, ethylene oxide, carboplatin, or cisplatin. As expected, chemical mutagens caused a clear-cut increase of the revertants in the Ames test. However, neither the static fields nor a combination of a static magnetic field with the time-varying bipolar gradient field or a pulsed high-frequency magnetic field caused an alteration in the number of revertants in the Ames test. No co-mutagenic effect of any magnetic field combination was observed. In conclusion, magnetic fields used during clinical magnetic resonance imaging (MRI) were neither mutagenic nor co-mutagenic.

Animals↗

PubMed2001

Sequestration of biological reactive intermediates by trapping as covalent enzyme-intermediate complex.

One important class of biological reactive intermediates arising in the course of human xenobiotic metabolism are arene and alkene oxides. The major safeguard against the potential genotoxic effects of these compounds is the microsomal epoxide hydrolase (mEH). This enzyme has a broad substrate specificity but--on the first sight--seems to be inadequately suited for this protection task due to its low turnover number with most of its substrates. The recent progress in the understanding of the mechanism of enzymatic epoxide hydrolysis has shed new light on this apparent dilemma: Epoxide hydrolases convert their substrates via the intermediate formation of a covalent enzyme-substrate complex, and it has been shown that the formation of the intermediate proceeds by orders of magnitudes faster than the subsequent hydrolysis, i.e. the formation of the terminal product. Thus, the enzyme acts like a molecular sponge by binding and inactivating the dangerous metabolite very fast while the subsequent product release is considerably slower, and quantification of the latter heavily underestimates the speed of detoxification. Usually, the slow enzyme regeneration does not pose a problem, since the mEH is highly abundant in human liver, the organ with the highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme is crucial for the control of the steady-state level of a substrate epoxide and can keep it extremely low. Once the mEH is titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to a sudden burst of the genotoxic effect. This prediction of the computer simulation is in perfect agreement with our experimental work. V79 Chinese Hamster cells that we have genetically engineered to express human mEH at about the same level as that observed in human liver are well protected from any measurable genotoxic effect of the model compound styrene oxide (STO) up to an apparent threshold level of 100 microM in the cell culture medium. In V79 cells that do not express mEH, STO triggers the formation of DNA strand breaks in a dose-dependent manner with no apparent threshold. Above 100 microM, the genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line.

Animals↗

PubMed2001

Long-time expression of DNA repair enzymes MGMT and APE in human peripheral blood mononuclear cells.

The DNA repair enzymes O6-methylguanine-DNA methyltransferase (MGMT) and apurinic/apyrimidinic endonuclease (APE, also known as Ref-1) play an important role in cellular defense against the mutagenic and carcinogenic effects of DNA-damaging agents. Cells with low enzyme activity are more sensitive to induced DNA damage and may confer a higher carcinogenic risk to the individuals in question. To study the level of variability of MGMT and APE expression in human, we analyzed in a long-time study MGMT and APE expression in peripheral blood mononuclear cells (PBMC) from healthy individuals. The data revealed high inter- and intraindividual variability of MGMT but not of APE. For MGMT, the interindividual levels ranged from 27 to 204 fmol/10(6) cells (7.6-fold, 40 healthy individuals). The intraindividual variation was determined by measuring MGMT repeatedly over 42 days, and was found to vary from 1.4-fold to 3.5-fold. Averaging over the measurement period, some individuals displayed low MGMT activity compared to others. In contrast, APE expression showed only a 2.9-fold difference between individuals and a 1.2 to 2.3-fold intra-individual long-time variation, and thus was less variable than MGMT. MGMT and APE expression were not correlated. Overall the results showed variable MGMT and rather constant APE levels in PBMC of healthy individuals measured over a long period.

Adult↗

PubMed2001

Gamma-glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines.

Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.

Aminopeptidases↗