Differential influence of the lipid peroxidation product 4-hydroxynonenal on the growth of human lymphatic leukaemia cells and human periopherial blood lymphocytes.

The purpose of this study was to compare the influence of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids, on the proliferation of malignant CEM-NKR lymphatic leukaemia cells and normal human lymphocytes (HPBM). Furthermore the growth modulating effect of phytohemagglutinin (PHA) on both cell lines was examined. The effects of HNE were monitored 18 hours and 3 days after incubation, using two different DNA-synthesis assays ([3H]-thymidine- and BrdU- incorporation) and a mitochondrial dehydrogenases activity assay (MTT). On the one hand HNE showed dose-dependent effects on both of the cell lines, while on the other hand a clear difference between the response of CEM-NKR cells and HPBM cells, respectively, to HNE was observed. On CDM-NKR cells, both concentrations of HNE caused significant cytotoxic effects on DNA-synthesis as well as on mitochondrial activity, while in contrast, HNE did not show any significant toxicity to HPBM cells. After 3 days there was even a slight stimulation of DNA synthesis with the physiological concentration of HNE. Furthermore the presence of PHA in the culture medium increased the difference of the response of CEM-NKR and HPBM cells, respectively to HNE.

Interheavy disulfide bridge in immunoglobulin G (IgG) reacting with dithionitrobenzoate. A unique feature in serum proteins.

Immunoglobulin G (IgG) of many species contains 'labile' disulfide bonds (SS*), which within 24 h undergo a disulfide exchange with dithionitrobenzoic acid (NBSSBN). Aims of the present study were to detect directly this type of SS* groups by means of 14C-labelled NBSSBN, and to investigate its possible presence in other serum proteins. NBSSBN reacts during the first 30 min of incubation with free SH groups, and thereafter with the sulfur atoms of SS* groups. The latter reaction reaches equilibrium after 24 h. The total of thionitrobenzoate residues (NBS) bound to IgG is called 'sigma S' and represents both SH and SS*. The results can be summarized as follows: (1) The measurement of the binding of 14C-NBSSBN gave identical sigma S values with IgG from humans and mice, as compared to the detection with the unlabelled reagent, which is based on the photometric determination of liberated NBS anions; whereas with IgG from rats some differences were observed which were ascribed to different batches of animals investigated; (2) experiments with electrophoretically separated serum proteins revealed only the gamma-globulins strongly binding 14C-NBSSBN in addition to the 30 min reaction, which indicates that SS* is confined to the gamma-globulin fraction; and (3) the significant decrease of sigma S in association with malignant tumors in man and animal models, which was previously described to be due to a specific alteration of the IgG subclass pattern, was likewise detected with the radiometrical method. Previous studies have identified SS* as one of the two inter-heavy disulfide bridges in IgG1, and possible implications of this group in specific functions of IgG1 are discussed.

Effect of oxidative stress by iron on 4-hydroxynonenal formation and proliferative activity in hepatomas of different degrees of differentiation.

It has been shown previously that oxidative stress by ferrous iron in vitro leads to an inhibition of proliferation of murine ascites tumour cells in vivo. This effect is associated with increased lipid peroxidation in terms of formation of the highly reactive aldehyde 4-hydroxynonenal (HNE), which has been shown to inhibit the proliferation of numerous tumours and to induce differentiation. It was the purpose of this article to study the occurrence and metabolism of HNE and its inducibility by oxidative stress in hepatomas of different degrees of differentiation to find further evidence for a possible role of HNE in proliferation and/or differentiation, because it is known that in hepatoma cells with a very low degree of differentiation basal lipid peroxidation is hardly detectable, while in normal hepatocytes the basal level of thiobarbituric acid reactive substances (TBArS) is rather high. MH1C1 hepatoma cells and Yoshida AH-130 hepatoma cells were chosen as highly differentiated and poorly differentiated tumour cells, respectively, and rat hepatocytes served as a control for normal liver phenotype. Ferrous histidinate (Fe/His) did not have a cytotoxic effect on Yoshida and MH1C1 cells, as measured by the LDH release test. In cell culture studies Fe/His revealed a dose dependent inhibition of the proliferation of Yoshida cells. The incorporation of 3H-thymidine into DNA of these cells was also inhibited by Fe/His in a dose-dependent manner, while the precursor uptake into the cytoplasm was unaffected. The basal levels of HNE were in the order: hepatocytes > MH1C1 cells > Yoshida cells. Both hepatocytes and Yoshida cells responded to the presence of Fe/His with increased formation of TBArS. Compared with hepatocytes the response of the Yoshida cells was greatly reduced. The response of cells to Fe/His with respect to HNE formation was decreased in the order: hepatocytes > MH1C1 cells > Yoshida cells, but in this case the differences were not very pronounced. The metabolic capacity of the cells to consume HNE was also decreased in the order: hepatocytes > MH1C1 cells > Yoshida cells. In this case the differences were very pronounced. These findings support the view that Yoshida cells with a low degree of differentiation and a low basal level of HNE are released from an inhibitory effect of HNE operative in hepatocytes and that HNE is causally involved in the iron induced inhibition of proliferation of poorly differentiated hepatoma cells.

The effect of reduced physical activity on longevity of mice.

Reduced physical activity leads, in female mice, to a reduction of the average and maximal life span. The average age at death of the inactive experimental group was 497 +/- 121 days (mean +/- S.D.) compared to 557 +/- 139 days in the active control group, and the six oldest inactive experimental mice died at age 732 +/- 50 days, while the six oldest active control mice died at 890 +/- 52 days. The restriction of mobility was connected with a higher growth rate and a higher body weight in spite of a significant decrease in food intake. In spite of a reduced food intake leading to a reduced whole body metabolism, the results show that mobility restriction shortens life span in female mice.

A decrease in reactive disulfide bonds of serum IgG signals a characteristic change in the IgG subclass patterns of rats bearing experimental tumors.

Previous studies have shown that human IgG1 contains a 'reactive' disulfide bridge (SS*), detectable by a 24-hour disulfide exchange reaction, and that the serum level of this IgG subclass is selectively diminished in patients with various malignant diseases. Here we present evidence that in rats IgG2b is the only subclass that carries one SS* per molecule. Furthermore, it is shown that rats inoculated with experimental tumor lines, i.e., the Yoshida hepatoma ascites tumor and the Walker 256 carcinosarcoma growing in ascites or as solid tumor, exhibit significantly decreased SS* per mole IgG which corresponds to a selective diminution of IgG2b. Although at later stages there is a quantitative correlation with the tumor burden, with the Walker tumor this effect becomes significant as early as 24 h after inoculation, i.e., well before exponential tumor growth and an absolute reduction of total IgG. Control animals injected intraperitoneally with either viable spleen cells or irradiated Walker 256 cells did not show comparable alterations in their IgG subclass profile. Thus, the selective defect of IgG2b requires the presence of viable and proliferating tumor cells. Possible mechanism(s) of tumor-associated shifts in IgG subclasses are discussed.

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

The 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 degrees 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 0.1 mM respectively. 4-Hydroxynonenal, in contrast, showed a continuous increase up to 41.1 nM (corresponding to 0.58 pmol/10(9) 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.

The inhibitory effect of 4-hydroxy-nonenal on DNA-polymerases alpha and beta from rat liver and rapidly dividing Yoshida ascites hepatoma.

The purpose of this study was to determine firstly whether the isolated enzyme DNA polymerase alpha, which functions within the DNA replicase system, exhibits different sensitivity against the thiol-blocking agent 4-hydroxy-nonenal (HNE) when adult rat liver and the rapidly dividing Yoshida ascites hepatoma were used as enzyme sources and, secondly, whether the reaction catalysed by DNA polymerase is the most sensitive step of the DNA replicase system of native cells. DNA polymerase alpha as well as the non-replicative DNA polymerase beta, isolated from both sources, were remarkably similar with regard to their sensitivity against HNE, as indicated by the incorporation of radioactive label from [3H]deoxy-thymidine-triphosphate into DNA. The transport of [14C]thymidine through the plasma membrane and the incorporation of this precursor into DNA were studied with neonatal hepatocytes and with hepatoma cells. The incorporation of thymidine was inhibited at lower concentrations of HNE in both cell lines than the transport process and the reaction catalysed by DNA polymerase alpha. It was concluded that in the DNA replicase system of native liver and hepatoma cells another process different from the reaction catalysed by DNA polymerase alpha is more sensitive to HNE.

[Fluorodensitometric determination of cytostatic acting Michael-adducts of alpha, beta-unsaturated aldehydes in biologic material]. / Fluorodensitometrische Bestimmung von zytostatisch wirksamen Michael-Addukten alpha, beta-ungesättigter Aldehyde in biologischem Material.

Alpha, beta-unsaturated aldehydes produce a selective cytostatic effect on tumor cells. By the formation of Michael adducts with cysteine, toxicity can be greatly reduced without impairing cytostatic effectiveness. To further enhance the selectivity of the toxic effect, it is necessary to be able to follow the agent's kinetics in the animal body. Among the analytic methods developed to this end, this paper represents the fluorescence derivatisation as a sensitive method for the determination of the Michael adducts of alpha, beta-unsaturated aldehydes in pharmaceutical preparations and in biological material. It is based on the reaction of the carbonyl groups with dansyl hydrazine. Determination is carried out by a combination of thin-layer chromatography with subsequent fluorodensitometric evaluation. The detection limit in blood is about 20 micrograms/ml. The relative standard deviation of this procedure ranges between 3 and 7%.

[Correlation between protein thiols and the doubling time of various rat hepatomas]. / Zusammenhang zwischen den Protein-Thiolen und der Dopplungszeit verschiedener Ratten-Hepatome.

The 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 less than 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.

[Intracellular effect of hydroxyalkenals on animal tumors (author's transl)]. / Intracelluläre Wirkung von Hydroxyalkenalen auf tierische Tumoren.

When incubated for 30 min in vitro, 4-hydroxyalkenals in a 5 x 10-3 M solution react with SH-groups of soluble cytoplasmic and nuclear proteins (ProtL-SH) of Morris hepatomas 9618 A and 5123 tc, and of Ehrlich ascites tumor (EAT) cells. The extent of the reaction strongly increases with decreasing doubling time of the respective tumors. Former experiments with EAT cells have shown that the reaction with cytoplasmic ProtL-SH causes inhibition of respiration, glycolysis, and probably of other SH-controlled processes associated with cell division. The intranuclear reaction leads to an inhibition of DNA- and RNA-synthesis. In a 2 x 10-4 M solution of hydroxyalkenals, however, the reaction with cytoplasmic ProtL-SH diminishes almost completely so that no measurable inhibition of respiration and only 3% inhibition of glycolysis are observed, while 20 to 30 percent of the nuclear ProtL-SH are still blocked. This corresponds well with a previous observation that a 2 x 10-4 M solution of hydroxypentenal inhibits DNA-synthesis by 90 percent.

Tumor host relations. V. Nitrogen metabolism in Yoshida sarcoma-bearing rats. Reduction of growth rate and increase of survival time by administration of physiological doses of branched-chain amino acids.

The continuous administration of physiological doses of the branched-chain amino acids leucine, isoleucine, and valine (Leu-Ile-Val) to Yoshida sarcoma-bearing rats caused a significant increase in the survival time by 32% and a significant reduction of tumor size after 3 weeks of growth by 33%. The shift of the nitrogen balance to negative values during the cachectic stage was delayed but not prevented. On the average, less nitrogen (-47 mg/day) were lost by Leu-Ile-Val treated rats compared with untreated tumor-bearing animals (-91 mg N/day). It appeared that Leu-Ile-Val increased the synthesis of carcass proteins, while it left the proteolysis rate unchanged, since the excretion of urea and creatinine was unaffected by these amino acids. The daily excretion of alpha-ketoglutarate, which is correlated with tumor size during the early stage of growth, was decreased during the first 2 weeks by Leu-Ile-Val, but remained for a longer period on a high level than in untreated tumor bearers. The results point to an improvement of the metabolic resistance against carcass protein depletion of the tumor-bearing host by the administration of branched-chain amino acids.

Tumor host relations. IV. Tissue distribution of citric acid cycle intermediates and of glutamate in tumor-bearing rats high level of alpha-ketoglutarate in solid Yoshida sarcomas.

To elucidate the origin of increased concentrations of alpha-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 cycle determines the concentration of KG in the tumor.