Helmward Zollner

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.

Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic activity, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.

Reaction of Glutathione with Conjugated Carbonyls

Abstract 1. GSH reacts with conjugated carbonyls according to the equation: G SH+R-CH=CH-COR⇆R-CH(SG)-CH2-COR. The forward reaction follows second order, the reverse reaction first order kinetics. It is assumed that this reaction reflects best the ability of conjugated carbonyls to inactivate SH groups in biological systems. 2. The rate of forward reaction increases with pH approx. parallel with αSH. Besides OH- ions also proton donors (e. g. buffers) increase the rate. The catalytic effect of pH and buffer is inter preted in view of the reaction mechanism. 3. The equilibrium constants as well as the rate constants for forward (k1) and reverse reaction show an extreme variation depending on the carbonyl structure. Acrolein and methyl vinyl ketone (kt = 120 and 32 mol-1 sec-1 , resp.) react more rapidly than any other carbonyl to give very stable adducts (half-lives for reverse reaction 4.6 and 60.7 days, resp.). Somewhat less reactive are 4-hydroxy-2-alkenals and 4-ketopentenoic acid (k1 between 1 and 3 mol-1 sec-1), but they also form very stable adducts showing half-lives between 3.4 and 19 days. All other carbonyl studied react either very slowly (e. g. citral, ethly crotonate, mesityl oxide, acrylic acid) or form very labile adducts (crotonal, pentenal, hexenal, 3-methyl-butenone). Comparing biological activities of con jugated carbonyls their reactivity towards HS (k1) and the stability of the adducts must be considered.

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.

Inhibition of some mitochondrial functions by acrolein and methylvinylketone.

Abstract The effects of acrolein and methylvinylketone on some mitochondrial processes have been studied. The respiration with various substrates is inhibited. The sensitivity to acrolein decreases in the following order: ADP-stimulated respiration with glutamate, DNP-stimulated respiration with glutamate, ADP-stimulated respiration with succinate, DNP-stimulated respiration with succinate and respiration with NADH of aged mitochondria. It is suggested that acrolein acts on three different sites; glutamate transport, Pi transport and succinodehydrogenase. The effect on Pi transport is competitive with respect to Pi. Methylvinylketone shows the same effects as acrolein, however, it is less effective by one order of magnitude.

Formation and Metabolism of the Lipid Peroxidation Product 4-Hydroxynonenal in Liver and Small Intestine

During reoxygenation of isolated rat hepatocytes or rat small intestine following 60 min of anoxia/ischemia an increase of the concentration of the cytotoxic lipid peroxidation product 4-hydroxynonenal was observed. This increase was short-termed. The rapid disappearance of 4-hydroxynonenal is due to its immediate reactions with cellular compounds and its metabolism. The capacity of eucaryotic cells to metabolize 4-hydroxynonenal was investigated. As primary and secondary metabolic products the glutathione-HNE conjugate, the 4-hydroxynonenoic acid, the corresponding alcohol 1,4-dihydroxynonene, the glutathione-dihydroxynonene conjugate and water were identified. It is postulated that the rapid metabolism of 4-hydroxynonenal and other aldehydic products of lipid peroxidation could be an important part of the intracellular antioxidative defense system.

The metabolism of 4-hydroxynonenal, a lipid peroxidation product, is dependent on tumor age in Ehrlich mouse ascites cells

4-Hydroxynonenal is a major product formed by lipid peroxidation from omega 6-polyunsaturated fatty acids as linoleic acid and arachidonic acid. This aldehyde is cytotoxic at high concentrations (in the range of 100 microM), disturbs cell proliferation at low concentrations and exhibits genotoxic effects. Furthermore, in the submicromolar range 4-hydroxynonenal is chemotactic and stimulates phospholipase C. 4-Hydroxynonenal is rapidly metabolized in eucaryotic cells. Here the metabolism of 4-hydroxynonenal was studied in suspensions of Ehrlich mouse ascites cells at different periods of the tumor age. The Ehrlich ascites tumor is a convenient biological model for the investigation of tumor cells in different age and proliferation phases of the tumor. The main products of 4-hydroxynonenal which were identified in the Ehrlich ascites cells were glutathione-HNE-conjugate, hydroxynonenoic acid and 1,4-dihydroxynonene. The formation of glutathione conjugates following the addition of 4-hydroxynonenal was higher in cells of the early phase in comparison with cells of the late phase of tumor growth. That was in accordance with the increased consumption of the reduced form of glutathione during 4-hydroxynonenal utilization. The degradation of 4-hydroxynonenal and other aldehydic products of lipid peroxidation is postulated to be an important part of the intracellular antioxidative defense system.

Therapieversuche mit Hydroxypentenal — II

The paper deals with the therapeutical effect of hydroxypentenal (HPE) on 3- and 8-day-old solid sarcoma 180 of mice. Subcutaneous peritumoral application of 4 · 10−7 m/g body weight given on the 4th and 7th, 9th and 12th day post implant., respectively, leads to a complete regression in 30%, considerable inhibition in 59% and uninfluenced growth in 11% of the three-day-tumour. The efficiency-index of 0.19 points to a significant and good tumour inhibition. In case of the eight-day-tumour, complete regression is observed in 8%, mediocre inhibition in 73%, uninfluenced growth in 19%. Efficiency-index 0.38. Observation time 21 days. Über die therapeutische Wirkung von Hydroxypentenal (HPE) auf 3 Tage und 8 Tage altes Sarkom 180 solid der Maus wird berichtet. Bei subcutaner peritumoraler Applikation von 4 · 10−7 Mol/g Körpergewicht am 4. und 7. Tag bzw. am 9. und 12. Tag post implant. wurde der 3 Tage alte Tumor in 30% der Fälle makroskopisch vollständig vernichtet, in 59% das Wachstum weitgehend verzögert und in 11% nicht beeinflußt. Der Wirkungs-Index von 0,19 entspricht einer deutlichen und guten Tumorhemmung. Ungünstigere Werte erbringt die Therapie des 8 Tage alten Tumors. Bei 8% der Tiere wurde der Tumor vollständig verhindert, bei 73% verzögert und bei 19% nicht beeinflußt. Wirkungs-Index 0,38, Beobachtungsdauer 21 Tage.

023 Qualitative and quantitative determination of metabolites of the lipid peroxidation product 4-hydroxynonenal from hepatocytes, enterocytes and tumor cells

During the degradation of lipid hydroperoxides as primary products of free radical-initiated lipid peroxidation aldehydic compounds are generated. 4-Hydroxynonenal (HNE) is a major aldehydic lipid peroxidation product which is formed from omega 6-PUFA. It exhibits cytotoxic, mutagenic, and genotoxic properties [1]. Increased levels of HNE were found in plasma and tissues under conditions of oxidative stress [ 2 4]. HNE is rapidly metabolized in eucaryotic cells. The aim of this study was the investigation of the different pathways of HNE metabolism. That includes the development of sensitive analytical procedures for the qualitative and quantitative determination of metabolic HNE products.

Ornithine uptake by isolated hepatocytes and distribution within the cell

Uptake of ornithine by isolated hepatocytes and its distribution within the cell was investigated. Ornithine uptake was energy independent and exhibited a saturable and a nonsaturable component. The Km value of the saturable component was 1.3 mM. At an external ornithine concentration of 0.5 mM the rate of ornithine uptake was 127 +/- 19 nmol/g. Lysine inhibited ornithine uptake, indicating the existence of an ornithine transport system. It was concluded that ornithine transport can limit urea synthesis in the state of transition from a low ammonia to a high ammonia supply.

Zusammenhang zwischen chemischer Konstitution und therapeutischer Wirkung α, β-ungesättigter Aldehyde am Ehrlich-Solid-Tumor der Maus

Biochemical and biological effects and chemical properties of three alpha,beta-unsaturated aldehydes [acrolein (I), 4-hydroxypentenal (II), and 4-hydroxyundecenal (III)] were compared. These three substances inhibited the incorporation of -3H-thymidine in Ehrlich-ascites-tumor cells in vitro in the following order: I is greater than III is greater than II. For reactivities with glutathion in vitro the order was: I is greater than II is greater than III is greater than. We concluded there from that the high activity of I, as to its inhibition of thymidine incorporation and its toxicity, was related to its high activity against SH-groups. With respect to therapeutic effects and hydrophilia the following order: II is greater than I is greater than III was observed. In our opinion the high hydrophilia of II is responsible for the significant and good inhibition of tumour growth.Biochemical and biological effects and chemical properties of three α, β-unsaturated aldehydes [acrolein (I), 4-hydroxypentenal (II), and 4-hydroxyundecenal (III)] were compared. These three substances inhibited the incorporation of 3 H-thymidine in Ehrlich-ascites-tumor cells in vitro in the following order: I ≫III>II. For reactivities with glutathion in vitro the order was: I≫II>III. We concluded there from that the high activity of I, as to its inhibition of thymidine incorporation and its toxicity, was related to its high activity against SH-groups. With respect to therapeutic effects and hydrophilia the following order: II≫I≫III was observed. In our opinion the high hydrophilia of II is responsible for the significant and good inhibition of tumour growth. Biochemische und biologische Wirkungen und chemische Eigenschaften dreier α, β-ungesättigter Aldehyde [Acrolein (I), 4-Hydroxypentenal (II) und 4-Hydroxyundecenal (III)] wurden verglichen. Hinsichtlich Hemmung des 3 H-Thymidin-Einbaues in Ehrlich-Ascites-Tumorzellen in vitro und Toxizität bei NMRI-Mäusen ergibt sich folgende Reihenfolge der Aktivitäten der drei Substanzen: I≫III>II. Hinsichtlich der Reaktivitäten gegenüber Glutathion in vitro ergibt sich I≫II>III. Daraus wird geschlossen, daß die besondes hohe Aktivität von I bezüglich der Hemmung des Thymidin-Einbaues und der Toxizität mit der besonders hohen Reaktivität gegenüber funktionellen SH-Gruppen zusammenhängt. Im Therapieeffekt auf den lokal infiltrierten Ehrlich-Solid-Tumor und in der Hydrophilie ergibt sich dagegen die Reihung II≫I≫III. Die höchste Hyydrophilie von II wird zur Erklärung der mit dieser Substanz festgestellten deutlichen und guten Tumorhemmung herangezogen.