Showing papers by "Franz Oesch published in 1992"

DNA strand breaks and DNA cross-links in peripheral mononuclear blood cells of ovarian cancer patients during chemotherapy with cyclophosphamide/carboplatin

Abstract: DNA strand breaks and DNA cross-links were detected in peripheral mononuclear blood cells of 15 ovarian carcinoma patients by alkaline filter elution. These patients received therapy with 600 mg/m2 of cyclophosphamide and 350 mg/m2 of carboplatin. Blood samples were taken a day before and 16 to 18 h after a therapy cycle. The patients showed an increased elution rate of 37% compared with that of healthy controls before the current cycle of chemotherapy, probably due to treatment in a previous cycle of therapy. The difference was statistically significant ( P < 0.02; U test). At the end of the actual cycle of therapy an average acceleration of the elution rate of 157% was found compared with that of controls ( P < 0.01; U test). Compared with the rate before the cycle of therapy, the mean elution rate after treatment was accelerated by 89% ( P < 0.01; Wilcoxon test). The amount of DNA-protein cross-links was also increased after drug application. The individual patients showed different responses after drug intake. While some patients showed hardly any alteration in the elution rate, others showed an acceleration of up to 400%. Monitoring the course of disease in six of these patients indicated that a strong acceleration in the elution rate after drug application is possibly linked to the success of the chosen cancer treatment as measured by a decrease in the tumor marker CA12-5 to the normal level. In another investigation the group of patients who had received non-alkylating antineoplastic agents showed no increase in DNA strand breaks compared with untreated controls. Thus, monitoring DNA single-strand breaks in the peripheral mononuclear blood cells of patients can help to evaluate the efficiencies of the cancer treatment as a composite of individual differences in resorption, metabolic activation and detoxification, and possibly some constitutional aspects of drug resistance to cyclophosphamide/cisplatin and probably to several other alkylating antineoplastic drugs. This may help in choosing an effective drug and in adjusting the doses of these drugs individually in the chemotherapy of cancer.

Dependency of the in vitro stabilization of differentiated functions in liver parenchymal cells on the type of cell line used for co-culture

Abstract: The differentiation status in cultures of primary rat liver parenchymal cells was determined by measuring the activities of various xenobiotic metabolizing enzymes. Most enzyme activities dropped rather rapidly in monocultures of parenchymal cells. The protein content and the activities of cytosolic epoxide hydrolase, glutathione S-transferase, andα-naphthol UDP-glucuronosyl transferase were, however, well stabilized in 7-day-old co-cultures of parenchymal cells with two different lines of rat liver nonparenchymal epithelial cells (NEC1 and NEC2). Phenol sulfotransferase and microsomal epoxide hydrolase activity were reduced in this coculture system after 7 days to about 30 and 20% of the initial activity. Generally, higher enzyme activities were measured in co-cultures with one specific epithelial cell line (NEC2) as compared to those with the other line (NEC1). C3H 10T1/2 mouse embryo fibroblasts supported the parenchymal cells even better than the two epithelial lines, because the activity of microsomal epoxide hydrolase was also stabilized. Glutathione transferase activity was increased over time in this co-culture system. Our results show that the differentiation status of liver parenchymal cells was much better stabilized in co-cultures than in monocultures but that, depending on the type of cells used for co-culture, great quantitative differences existed. The entire pattern of xenobiotic metabolizing enzyme activities could not be stabilized at the kind of levels found in freshly isolated parenchymal cells.

Persistence of the cholangiocellular and hepatocellular lesions observed in rats fed a choline-deficient/DL-ethionine-supplemented diet.

Abstract: Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine (CDE) for 4, 6, 10, 14 or 22 weeks followed by a standard diet for up to 59 weeks. Liver sections were histochemically analyzed for the following parameters: basophilia, glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The stop experiments revealed that many of the oval cells proliferating during the first 4-6 weeks may undergo necrotic changes and disappear with time, whereas cholangiofibroses appearing in animals fed CDE for at least 10 weeks are persistent lesions. The sequence of lesions seen in this study, leading from persistent oval cells through cholangiofibroses to cholangiofibromas, strongly suggests that the oval cells are the precursor cells of cholangiocellular tumors. The proliferating oval cells and the hepatic foci consisting of clear and acidophilic or mixed cell populations were always spatially separated and no transitions between oval and parenchymal cells were observed. These results argue against a precursor-product relationship between oval and parenchymal cells. Both proliferating and persistent oval cells, cholangiofibroses and cholangiofibromas showed a strong staining for G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT; low PHO, SYN and G6PASE activities were also detected in these lesions. Persistent glycogen-storage foci, which developed in all rats fed CDE for 4-14 weeks followed by a normal lab chow for over a year, had increased PHO, G6PDH, MDH, ALKPASE and GGT activities, while SYN, GAPDH and G3PDH activities remained unaltered and G6PASE activity decreased. Mixed cell foci appearing in animals fed CDE for 22 weeks followed by a normal lab chow for 59 weeks had strongly increased G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities as well as decreased G6PASE activity. These results indicate that the characteristic metabolic pattern of preneoplastic hepatic foci is independent of the further administration of the carcinogenic diet. The shift from glycogen metabolism to glycolysis and the pentose phosphate pathway occurring during the later stages of CDE-induced hepatocarcinogenesis is an autogenous process apparently directing the disturbed carbohydrate metabolism towards alternative metabolic pathways. A similar metabolic shift also seems to take place during cholangiocarcinogenesis.

Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases.

Abstract: 1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.

Glutathione conjugation of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene isomers by human glutathione transferases.

Abstract: Each of the four stereoisomers of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene [(+)- and (-)-anti-BPhDE and (+)- and (-)-syn-BPhDE] has been incubated with the human glutathione transferase (GST) isoenzymes GST A1-1, GST M1-1 and GST P1-1, representing class alpha, mu and pi respectively, and glutathione (GSH). The conjugates formed were analyzed by HPLC and the results demonstrate that all GST isoenzymes catalyze the formation of GSH conjugates of all BPhDE isomers. However, a marked variation in catalytic efficiencies was observed (0.122-1.28/mM/s). These values are considerably lower than those previously estimated for the bay-region diol epoxides of benzo[a]pyrene (B[a]P) and human GSTs. The (+)-syn and (-)-anti-BPhDE (1R,2S-epoxide absolute configuration) were in general better substrates than the corresponding 1S,2R-epoxides. In accordance with previous observations with the diolepoxides of B[a]P, GST P1-1 was highly selective towards the BPhDE isomer with 4R,3S-diol 2S,1R-epoxide absolute configuration, i.e. (-)-anti-BPhDE, whereas GST A1-1 and M1-1 preferentially catalyzed the conjugation of (+)-syn-BPhDE (4R,3S-diol 2R,1S-epoxide absolute configuration). Overall, the most active isoenzyme was GST A1-1. Analysis by NMR spectroscopy of the GSH conjugates of BPhDE demonstrate that the reaction with GSH generally takes place by trans-addition of the thiol group at the benzylic C-1 carbon. The low catalytic efficiencies of human GSTs with BPhDE as compared to diolepoxides of B[a]P may be explained in part by the more crowded bay-region and substantially lower chemical reactivity (e.g. delta Edeloc/beta) of the former compounds.

Applications of stable V79-derived cell lines expressing rat cytochromes P4501A1, 1A2, and 2B1

Abstract: 1. Chinese hamster V79-derived cell lines, stably expressing cytochromes P4501A1, 1A2, and 2B1 activities, were constructed by genetic engineering in continuation of our work to establish a battery of V79 derived cell lines designed to study the metabolism of xenobiotics.2. Cell lines XEM1 and XEM2, expressing cytochrome P4501A1, were capable of the O-dealkylation of 7-ethoxycoumarin and the hydroxylation of benzo[a]pyrene.3. Cell lines XEMd.MZ and XEMd.NH, expressing P4501A2, were shown to hydroxylate 17β-estradiol and 2-aminofluorene.4. Cell line SD1, expressing cytochrome P4502B1, was able to hydroxylate testosterone stereo- and regio-specifically at the 16α and 16β positions.5. Cell lines were validated in mutagenicity, cytotoxicity, and metabolism studies employing benzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, cyclophos-phamide, ifosfamide, and picene.6. Construction of metabolically-competent V79-derived cell lines be recombinant DNA technology will be a fundamental improvement for ...

Relevance of environmental alkylating agents to repair protein O6-alkylguanine-DNA alkyltransferase: determination of individual and collective repair capacities of O6-methylguanine.

Abstract: The repair capacity for O6-methylguanine was determined in cell homogenates of peripheral blood lymphocytes of 35 automobile industry workers, exposed to rubber and tires, and of 35 clinical workers, handling cancer chemotherapeutic agents, compared to control groups. lambda-Phage DNA containing one 32P-labeled O6-methylguanine in each BamHI site was used as substrate for the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). The clinical personnel showed in the mean a highly significantly (P = 0.0014, Wilcoxon U test, Mann and Whitney) reduced activity of the repair enzyme [3.28 +/- 0.28 (SEM) fmol AGT/micrograms DNA] as compared to 37 control persons (4.88 +/- 0.32 fmol AGT/micrograms DNA). The mean AGT value of the automobile industry workers (4.40 +/- 0.28 fmol AGT/micrograms DNA) was not significantly different (P = 0.1303) from the mean of 38 examined controls (5.00 +/- 0.28 fmol AGT/micrograms DNA). By dividing these employees into subgroups according to their different work environments (handling of rubber fittings, tire mounting, and tire storage, respectively) the mean AGT value of the 15 tire storage workers was significantly (P = 0.0270) lower (3.80 +/- 0.36 fmol AGT/micrograms DNA) than the mean value of the controls. The interindividual variations in the activity of AGT were 5.1- and 6.5-fold in the control groups and 5.6-fold for the automobile industry workers; the largest variations were found in the group of the clinical personnel with 12.6-fold. No significant correlations between AGT and age, sex, or smoking behavior were observed in any of the groups examined. The decrease in AGT activity will render the afflicted individuals more susceptible to further exposure to methylating agents.

Sequence of a novel cytochrome CYP2B cDNA coding for a protein which is expressed in a sebaceous gland, but not in the liver.

Abstract: The major phenobarbital-inducible rat hepatic cytochromes P-450, CYP2B1 and CYP2B2, are the paradigmatic members of a cytochrome P-450 gene subfamily that contains at least seven additional members. Specific oligonucleotide probes for these genomic members of the CYP2B subfamily were used to assess their tissue-specific expression. In Northern-blot analysis a probe specific to gene 4 (which is designated now as CYP2B12) hybridized to a single mRNA present in the preputial gland, an organ which is used as a model for sebaceous glands, but did not hybridize to mRNA isolated from the liver or from five other tissues of untreated or Aroclor 1254-treated rats. The cDNA sequence for the CYP2B12 RNA was determined from overlapping cDNA clones and contained a long open reading frame of 1476 bp. The nucleotide sequence of the CYP2B12 cDNA was 85% similar to the sequence of the CYP2B1 cDNA in its coding region and was different from any CYP2B cDNA characterized until now. The cDNA-derived primary structure of the CYP2B12 protein contains a signal sequence for its insertion into the endoplasmic reticulum and the putative haem-binding site characteristic of cytochromes P-450. A part of the potential haem pocket of CYP2B12 was identical with a similar structure in a bacterial protocatechuate dioxygenase. In immunoblot analysis of preputial-gland microsomes, antibodies against CYP2B1 recognized a single abundant protein with a lower apparent molecular mass than that of CYP2B1. Our results demonstrate that the CYP2B12 protein has the potential to be enzymically active and are the first demonstration that a member of the CYP2B subfamily is expressed exclusively and at high levels in an extrahepatic organ.

In vivo formation of aflatoxin B1-DNA adducts in parenchymal and non-parenchymal cells of rat liver.

Abstract: The induction of hepatocellular carcinoma from liver parenchymal cells in laboratory animals by aflatoxin B1 (AFB1) is well documented. In contrast no tumours arising from the sinusoidal cell population have been reported after exposure to AFB1. The apparent resistance of the latter cell type was investigated at the level of DNA adduct formation in vivo in male Sprague-Dawley rats. Liver parenchymal and non-parenchymal cell populations were isolated from rats at 20 min and 1, 24 and 72 h after administration of 240 microCi (0.6 mg) [G-3H]AFB1/kg. AFB1-DNA binding was observed in both liver cell subpopulations and was 3- to 5-fold higher in parenchymal cells than in non-parenchymal cells. The major DNA adduct found in parenchymal cells at 1 h after AFB1 administration was 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-gua), whereas at later time points the persistent secondary adduct, AFB1-formamidopyrimidine, predominated. In contrast, AFB1-gua was not observed at any time in DNA from non-parenchymal cells and the secondary adducts predominated throughout. These observations are discussed with reference to the susceptibility of different liver cell types to AFB1-carcinogenesis and the possible roles of the major AFB1-DNA adduct species.

Xenobiotic-metabolizing enzyme activities in hybrid cell lines established by fusion of primary rat liver parenchymal cells with hepatoma cells.

Abstract: 1. The activities of xenobiotic-metabolizing enzymes were determined in hybrid cell lines (hepatocytoma, HPCT) which have been established by fusion of liver parenchymal cells from adult rat (PC) with cells from a Reuber hepatoma cell line (FAO). 2. Cytochrome P450 was not measurable spectrophotometrically in FAO and HPCT. P450-dependent conversion of testosterone was below the detection limit in FAO and only marginally present in HPCT. 3. Microsomal and cytosolic epoxide hydrolase, glutathione S-transferase and phenol sulphotranserase were low or even below detection limit in FAO. These enzyme activities were significantly higher in HPCT and correspond to about 1-10% the activities measured in PC. 4. 1-Naphthol UPD-glucuronosyl transferase activity was about 20% in FAO and about 100% in HPCT compared to PC. 5. Metabolic conversion of benzo[a]pyrene was low in FAO, high in PC, and intermediate in HPCT. The presented data, however, do not allow the conclusion whether this intermediate rate is catalyzed by similar P450 isoenzymes as in PC. 6. Due to the easily measurable phase II-metabolizing enzyme activities HPCT may, however, be useful for in vitro enzyme induction or repression studies.

Contact-Inhibition of Growth by Complex Carbohydrates

Abstract: 形質転換を受けていない哺乳動物細胞の増殖の調節は細胞密度によって厳密な支配を受けている。細胞密度が増加すると、増殖は加速度的に抑制され、ついにはコンフルエントな状態で停止する。我々はコンタクトインヒビンと言われる糖タンパクを形質膜から単離した。固定化されたコンタクトインヒビンはヒトの二倍体繊維芽細胞の増殖の接触依存性阻害に関与している。これは培養皿で疎に成育している細胞の増殖を、可逆的で且つ毒とは異なる様式でピコモルのレベルで阻害するが、一方、可溶化された状態では活性がない。生物学的に活性な部位は専らN-結合型糖鎖に存在している。更に効果的に増殖を阻害するにはN-結合型糖鎖の末端にβ-結合したガラクトース残基が存在しなければならない。抗コンタクトインヒビン抗体の存在下でヒト繊維芽細胞を培養することによって、細胞は2倍の飽和密度に達することで増殖し、同時に広範囲な複雑に十字交差した細胞とフォーカスの出現を伴う。コンタクトインヒビンは形質膜レセプターと特異的に相互作用し、その結合活性はリン酸化の度合にようて制御されている。癌の無制限の増殖はコンタクトインヒビンレセプターの過度のリン酸化に起因していることが示唆されている。

Chemotactic migration of human diploid fibroblasts is inhibited by contactinhibin.

Abstract: Dear Editor: Cellular migration is required for a variety of biological processes such as organ formation or tissue remedeling after tissue injury. In order to function as a controlled process, at least two prerequisites have to be fulfilled: i) migration must be directional and it) migration must be stopped when the original size of a tissue has been achieved. Directional migration or chemotaxis is induced by well defined chemoattractants such as lymphokines (7), collagenous peptides (8), fibronectin (3) and PDGF (9). In addition, only cells directly facing a free space or a wound are allowed to respond to chemoattraetants with migration, while cells surrounded by neighboring cells are migration inhibited (1,2,6). After rebuilding injured tissue by invasion and division of cells, a negative migration--and proliferation signal must be generated. It has long been accepted that cell-cell contacts are required for a stop of migration, although the molecular basis has since not yet been identified. We have shown previously that contactinhihin, a 60-70 kDa plasma membrane glycoprotein is responsible for contact-dependent inhibition of growth (11). Therefore, experiments were undertaken to address the question whether contactinhibin might also be involved in the inhibition of chemotactic migration. Table 1 shows, that the degree of chemotactic migration of human fibroblasts was inversely correlated to the cell density seeded in the upper compartment of the Boyden chamber. At 2 × 105 cells seeded onto the filter, the cell density most commonly used in similar studies, approximately 17% of the cells have migrated after 4 hours. This is in good agreement with experiments where human fibroblasts have been used (9). Chemically transformed mouse fibro-

Stable Expression of Heterologous Microsomal Epoxide Hydrolase in BHK21 Cells: Influence on the Mutagenicity of Benzo[a]pyrene 4,5-Oxide

Abstract: Most environmental mutagens and carcinogens require metabolic activation to electro- philic intermediates capable of reacting with cellular target structures, such as DNA. These electrophilic intermediates are in addition subject to metabolic detoxification. This metabolism is mainly controlled by enzymes whose expression is very variable. Among other things, various enzymes are inducible by environmental chemicals. Understanding the toxicology of chemicals (for example, species differences, idiosyncrasias, organotropisms) therefore requires knowledge of critical host factors. One approach towards this goal involves the use of purified enzymes in metabolism and toxicological studies (Glatt et al., 1983; Glatt and Oesch, 1986). This approach, however, is laborious, the purification may alter enzymatic properties, and it may be difficult to rule out the influence of impurities in the enzyme preparation, especially when large families of structurally closely related enzymes exist, as is common in xenobiotic metabolism.

DNA strand break induction, mutagenicity, and cytotoxicity of the mycotoxins 11-β-hydroxy-7-deoxy-rosenonolactone, rosenonolactone, and trichothecin.

Abstract: 11-β-hydroxy-7-deoxy-rosenonolactone (TSS1), a mycotoxin of the rosenane class, was tested on cytotoxicity, induction of DNA single strand breaks and muta-genicity. Its effects were compared to those of rosenonolactone and trichothecin. TSS1 had stronger antibiotic activity againstEscherichia coli (EC 50: 10μg/mL) than rosenonolactone (EC 50: >200μg/mL) but weaker activity than trichothecin (EC 50: 3μg/mL). The same order of activity was found for the inhibition of yeast fermentation (EC 50 of TSS1: 45μg/mL; EC 50 of rosenonolactone: > 120μg/mL; EC 50 of trichothecin: 3.4μg/mL). In the trypan blue exclusion test using V79 Chinese hamster cells, TSS1 proved to be cytotoxic (EC50: 30μg/mL) at even lower doses than trichothecin (EC50: 200μg/mL). Rosenonolactone had no significant toxicity up to the highest soluble concentration (500μg/mL). DNA single strand breaks caused by TSS1 occurred at the same concentrations at which damage of the cell membrane became apparent. For trichothecin single strand breaks were detected only at concentrations at which the membrane was already highly damaged. No single strand breaks were observed in V79 cells after incubation with rosenonolactone up to the limit of solubility (500μg/mL). In the reversion assay withhis Salmonella typhimurium strains TA 98 and TA 100, no mutagenicity was observed for any of the examined mycotoxins up to 800μg/plate with and without the addition of a rat liver preparation for metabolism of the test compound.