H. M. Tillian

Iron-induced lipid peroxidation and inhibition of proliferation in Ehrlich ascites tumor cells

SummaryThe purpose of this study was to find further experimental evidence for the postulated negative association between the extent of lipid peroxidation in tumor cells and their proliferative behavior. After incubation of Ehrlich ascites tumor cells at 37°C for 30 min with increasing concentrations of Fe(II) histidinate (Fe/His) the following parameters were determined: the formation of lipid hydroperoxides was measured fluorimetrically after reaction with dichlorofluorescein; 4-hydroxynonenal was determined by reversed-phase high-pressure chromatography after derivatization with dinitrophenylhydrazine; as a third parameter of lipid peroxidation the formation of 2-thiobarbituric-acid-reactive substances was determined. The proliferative activity was determined by measuring the growth rate in vivo after reimplantation i.p. of the tumor cells into mice. Trypan-blue exclusion tests for viability were performed before reimplantation. The reliability of the trypan-blue exclusion tests was checked by comparing the results with another parameter of viability, the release of the cytosolic enzyme lactate dehydrogenase. The concentration both of lipid hydroperoxides and of 2-thiobarbituric-acid-reactive substances showed a biphasic dependence on the concentration of Fe/His with maximal increase at iron concentrations of 0.25 mM and 0.1mM respectively. 4-Hydroxynonenal, in contrast, showed a continuous increase up to 41.1 nM (corresponding to 0.58 pmol/109 cells) with increasing iron concentration in the range from 0.1 mM to 0.6 mM. The total number of tumor cells, when determined 5 days after reimplantation, continuously decreased with increasing iron concentration, showing half-maximal inhibition at about 0.22 mM Fe. The exclusion of the trypan-blue dye was unaffected by the presence of iron at any concentration used. Similarly, iron had no influence on the release of lactate dehydrogenase. The results support the hypothesis that 4-hydroxynonenal may act as an inhibiting messenger between endogenic lipid peroxidation and proliferation.

Tumor host relations. II. Increase of alpha-ketoglutarate in whole blood and urine and hypoalbuminemia of rats bearing the solid rhabdomyosarcoma BA 1112 and the ascitic or solid forms of Walker-carcinoma 256 and Yoshida-sarcoma.

SummaryReferring to the increased α-ketoglutarate (I) concentration in blood of cancer-bearing humans, the concentration of (I) in blood and of albumin (II) in serum were measured in 85 tumor-bearing and control rats by fluorimetry and cellulose acetate electrophoresis, respectively. (I) was elevated on the average by a factor of 1.82 and (II) was decreased by a factor of 0.73 in rats bearing the solid Rhabdomyosarcoma BA 1112 and the ascitic or solid forms of Walker-carcinoma 256 and Yoshida-sarcoma compared with tumor-free animals. All deviations were found to be statistically significant for the different tumor types, with the exception of the increase of (I) in Walker-ascites tumor-bearing rats.For rats bearing the ascitic and solid Yoshida-sarcoma and the ascitic Walker-carcinoma 256 and for tumor-free rats a negative correlation could be observed between increase of (I) and decrease of (II).The increase of (I) in blood was paralleled by an increased excretion rate of (I) into urine. In rats bearing the solid Yoshida-sarcoma the daily excreted amount of (I) was significantly increased by a factor of 1.86 compared with tumor-free animals. The excretion of (I) was already increased in the first week after tranplantation, remained constant for two more weeks and decreased sharply below normal values prior to death.The results suggest that rats bearing the inquested tumors are suitable models for metabolic studies on causes of pathological concentrations of (I) in blood of cancer patients.

Zusammenhang zwischen den Protein-Thiolen und der Dopplungszeit verschiedener Ratten-Hepatome

SummaryThe SH content of the soluble proteins (nanomol./mg protein) from five transplantable rat hepatomas and from the DENA-hepatoma were determined with dithionitrobenzoate (Ellman reagent). Both the total number of thiols as well as the number of SH groups that can be blocked by hydroxypentenal (HPE) increase with increasing growth rate of the tumors. In comparison with the protein thiol content of the slowest growing DENA-hepatoma (doubling time 100 days), the total protein thiols of the fastest growing Yoshida hepatoma (doubling time 2,5 days) increase by 100% and the HPE-sensitive protein thiols by 350%. The total protein thiols are significantly correlated with the growth rate (probability of error 5%), the HPE-sensitive thiols are correlated with high significance (probability of error <1%). These results are in accordance with the “Molecular Correlation Concept” of G. Weber and might possibly be understood as a consequence of reprogramming of gene expressions.ZusammenfassungDer SH-Gehalt der löslichen Proteine (nMole/mg Protein) von fünf transplantablen Ratten-Hepatomen und des DÄNA-Hepatoms wird mit Dithionitrobenzoat (Ellmans-Reagenz) bestimmt. Sowohl die gesamten Thiole als auch insbesondere die Zahl der SH-Gruppen, die mit Hydroxypentenal (HPE) blockiert werden, steigen mit zunehmender Wachstumsgeschwindigkeit der Tumoren an. Verglichen mit dem Thiolgehalt des am langsamsten wachsenden DÄNA-Hepatoms (Dopplungszeit ca. 100 Tage), steigen die gesamten Protein-Thiole des am schnellsten wachsenden Yoshida-Hepatoms (Dopplungszeit 2,5 Tage) um 100% an, die HPE-reaktiven Protein-Thiole dagegen um 350%. Die gesamten Protein-Thiole sind mit einer Irrtumswahrscheinlichkeit von 5% signifikant mit der Wachstumsgeschwindigkeit assoziiert, die HPE-reaktiven Thiole dagegen hochsignifikant (Irrtumswahrscheinlichkeit <1%). Diese Ergebnisse stehen in Übereinstimmung mit dem Konzept der molekularen Korrelationen von G. Weber und dürften danach als Folge einer “Re-Programmierung von Gen-Expressionen” zu verstehen sein.

Tumor host relations@@@Tumor - Wirt - Beziehungen: IV. Tissue distribution of citric acid cycle intermediates and of glutamate in tumor-bearing rats high level of ?-ketoglutarate in solid Yoshida sarcomas@@@IV. Gewebsverteilung von Citrat-Zyklus Metaboliten und von Glutamat in tumortragenden Ratten Hohe Konzentration von ?-Ketoglutarat in Soliden Yoshida Sarcomen

ZusammenfassungZur Untersuchung der Ursache der erhöhten α-Ketoglutarat (KG) Konzentration in Tumorträgern wurde die Konzentration des KG sowie der Metaboliten Citrat, Succinat, Malat und Glutamat in Tumor, Leber, Gastrocnemius-Muskel und Blut von tumorfreien und Ratten, die das solide Yoshida Sarcom trugen, untersucht.Die Summe dieser Metaboliten war in der Leber und im Blut von Tumorträgern signifikant erhöht gegenüber den entsprechenden Geweben gesunder Ratten Unter den einzelnen Metaboliten waren Glutamat und Malat in der Wirtsleber signifikant erhöht. Die absoluten Konzentrationen waren jeweils in der Wirtsleber am höchsten, mit Ausnahme des KG, dessen Konzentration im Tumor am höchsten war. Es wurde geschlossen, daß die Erhöhung der KG Konzentration im Blut von Tumorträgern hauptsächlich auf den Tumor selbst zurückzuführen ist.Im Gastrocnemius-Muskel wurden keine signifikanten Stoffwechselveränderungen beobachtet. Die Konzentration des KG in diesem Muskel sowohl von gesunden als auch von Wirtstieren korrelierte signifikant mit jener des Glutamats. Im Tumor korrelierte die KG Konzentration signifikant mit der Summe der Konzentrationen von Citrat, Succinat und Malat. Diese Art der Korrelation war in der Leber und im Muskel gesunder und tumortragender Tiere nicht gegeben. Weiteres wurde weder in der Leber noch im Tumor eine Korrelation zwischen KG und Glutamat beobachtet. Diese Ergebnisse sprechen dafür, daß der metabolische Fluß durch den Citrat-Zyklus bestimmend ist für die Konzentration des KG im Tumor.SummaryTo elucidate the origin of increased concentrations of α-ketoglutarate (KG) in tumor bearers the tissue distribution of KG together with the related metabolites citrate, succinate, malate, and glutamate was determined in tumor, liver, gastrocnemius muscle, and blood of rats bearing the solid Yoshida sarcoma and of tumor-free rats.The sum of these metabolites was significantly increased in host liver and blood, respectively, compared with the corresponding tissues of normal rats. Among single metabolites glutamate and malate were significantly increased in host liver. The absolute concentrations were highest in host liver with the exception of KG, which was highest in the tumor. This was taken as indicative for the tumor as the prime source of increased KG in blood of tumor-bearers. No significant metabolic deviations were found in gastrocnemius muscle. The concentration of KG in this muscle of both normal and host animals correlated significantly with that of glutamate.In the tumor the concentration of KG correlated significantly with that of citrate plus succinate plus malate. This type of correlation was absent in liver and muscle of both normal and host animals. Moreover, no correlation existed between KG and glutamate either in liver or in tumor. It was suggested that the metabolic flux through the citric acid cycle determines the concentration of KG in the tumor.