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ß-Carotene cleavage products induce oxidative stress in vitro by impairing mitochondrial respiration¹

WERNER SIEMS^*, OLAF SOMMERBURG, LORENZ SCHILD^#, WOLFGANG AUGUSTIN^#, CLAUS-DIETER LANGHANS and INGRID WISWEDEL^#2

^* Herzog-Julius Hospital for Rheumatology and Orthopedics Bad Harzburg;
^# Department of Pathological Biochemistry, Institute of Clinical Chemistry and Pathological Biochemistry, Otto-von-Guericke University Magdeburg;
University Children’s Hospital Heidelberg; and
University Children’s Hospital Ulm, Germany

²Correspondence: Department of Pathological Biochemistry, Institute of Clinical Chemistry and Pathological Biochemistry, Medical Faculty, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail: ingrid.wiswedel{at}medizin.uni-magdeburg.de

SPECIFIC AIMS

This study was undertaken to elucidate the role of mitochondria in increasing the risk of cancer after intake of carotenoids by individuals exposed to oxidative stress to an unusual extent (e.g., smokers and asbestos workers). We hypothesize that degradation products of ß-carotene, which are increasingly formed during heavy oxidative stress, contribute to the carcinogenic effects, as were found in the Alpha-Tocopherol, Beta-Carotene-Cancer-Prevention study and the Beta-Carotene and Retinol Efficacy Trial. We investigated whether carotenoid cleavage products (CCP)—namely, retinal, ß-ionone, and mixtures of cleavage products—which were generated in vitro under oxidative conditions increase oxidative stress in isolated rat liver mitochondria by impairing mitochondrial function.

PRINCIPAL FINDINGS

1. Carotenoid cleavage products inhibit oxidative phosphorylation
Carotenoid cleavage products were obtained by the reaction of ß-carotene, retinal, and ß-ionone with hypochlorous acid at room temperature. The generation in the presence of hypochlorite mimics the in vivo formation in inflammatory regions after activation of polymorphonuclear leukocytes. Retinal and ß-ionone are CCP, also used in the experiments. All CCP investigated strongly inhibited ADP-induced increase in respiration (state 3 minus state 4 respiration) in a dose-dependent manner (Fig. 1 ). The presence of 20 µM CCP led to a 30–50% decrease. Low concentrations of CCP, such as 1 µM and even 0.5 µM exerted significant inhibition of ADP-induced increase in respiration. The inhibition by retinal was 12.4 ± 0.5% at 1 µM and 6.3 ± 2.9% at a concentration of 0.5 µM. The inhibition range for the different CCP was between 5 and 12% at 1 µM and between 3 and 6% at 0.5 µM. State 4 respiration (respiration in the presence of hydrogen supplying substrates, but without ADP), was hardly affected, which rules out significant changes in membrane permeability. Uncoupled respiration (state 4u, after addition of FCCP) was not significantly affected by ß-carotene cleavage products (CP), retinal and retinalCP, and only to a small extent by ß-ionone and ß-ionone cleavage products in comparison to state 3 respiration. This documents that the capacity of the respiratory chain for respiration was not considerably influenced by CCP. Impairment of two candidates, namely, the F₀F₁-ATPase and/or the adenine nucleotide translocator, may be responsible for the decrease in respiration.

View larger version (22K): [in this window] [in a new window]   Figure 1. Influence of CCP on respiration. Rat liver mitochondria were incubated at 30°C in a medium containing 10 mM sucrose, 120 mM KCl, 15 mM NaCl, 20 mM TRIS, 2 mM MgCl2, 5 mM NaH2PO4, pH 7.4 (incubation medium). Five types of CCP were used: retinal, ß-ionone, mixtures of retinal (retinalCP) or ß-ionone (iononeCP), or ß-carotene cleavage products (caroteneCP). Concentrations of each CCP were 0.5 µM, 1 µM, 5 µM, or 20 µM, respectively. Inhibition of respiration is presented as a decrease in the difference of respiration after and before ADP addition (state 3 minus state 4 respiration) in % of complete inhibition. 100% inhibition corresponds to a decrease in respiration of 53.4 ± 3.5 nmol O2/mg/min, which is the ADP-induced increase in respiration of controls. Values are given as mean ± SE from 3 independent experiments.

ß-Carotene cleavage products and ß-ionone at concentrations of 20 µM, which inhibited ADP-induced increase in respiration by 40%, exerted no effect on the kinetics of cyanide-induced membrane depolarization. Since dissipation of the membrane potential depends on the activity of the F₀F₁-ATPase, this observation supports the suggestion that the inhibition of respiration by decomposition products of carotenoids is not caused by their interaction with this enzyme, but mainly by interaction with the adenine nucleotide translocator.

2. Carotenoid cleavage products induce depletion of glutathione and protein-SH and formation of malonic dialdehyde in mitochondria
The mitochondrial GSH content dramatically decreased in the presence of retinal and other ß-carotene cleavage products within 20 min of incubation, depicted in Fig. 2 . Most intense decreases were observed in the presence of retinal and retinal cleavage products. Both CCP at a concentration of 20 µM led to a GSH decrease from 6.21 ± 0.54 nmol/mg protein (control) to 1.71 ± 0.32 (retinal) and 2.51 ± 0.44 (retinalCP). Parallel to this, GSSG content increased. Twenty minutes incubation of mitochondria in the presence of 20 µM retinal led to a threefold increase in GSSG/GSH ratio compared to controls. CCP caused a decrease in total mitochondrial glutathione pool (GSH+GSSG), most likely due to GSSG efflux.

View larger version (23K): [in this window] [in a new window]   Figure 2. Decrease in mitochondrial GSH by carotenoid cleavage products. Rat liver mitochondria were incubated at 30°C for 20 min in the incubation medium in the presence of 20 µM CCP. Values (mean±SE) from 8 measurements (control and ß-caroteneCP) or 4 measurements (for retinal, retinalCP, ß-ionone, and ß-iononeCP) are presented.

Depletion of total protein-SH was found after incubation with each of the CCP. The mitochondrial protein-SH content decreased from 85.9 ± 4.6 nmol SH/mg protein (control) to 67.7 ± 1.9, and 78.2 ± 7.7 nmol SH/mg protein in the presence of 20 µM retinalCP and retinal, respectively. Thus, total protein-SH loss was markedly higher than the decrease in GSH.

The formation of malonic dialdehyde (MDA) was used to document lipid peroxidation after exposure of mitochondria to CCP. MDA levels were enhanced between 7.4- (ß-iononeCP) and 16.4-fold (ß-ionone) in relation to control incubations. However, the amount of MDA (up to 120 pmol/mg protein) was small compared to MDA concentrations formed during iron/ascorbate-induced lipid peroxidation in isolated mitochondria.

CONCLUSIONS

Besides enzymatic formation of CCP by dioxygenases, epoxidases, hydroxylases, dehydrogenases, and aldehyde oxidases, CCP can be formed nonenzymatically. The attack on carotenoids by different free radical species essentially results in the formation of numerous breakdown products. Among them are aldehydes, carbonyls, and epoxides. Retinal, ß-ionone, and short-chain cleavage derivatives of ß-carotene, retinal and ß-ionone formed nonenzymatically in the presence of high hypochlorite concentrations, were used in order to investigate their effects on mitochondrial energy metabolism. These cleavage products may be formed in vivo under different conditions, e.g., by polymorphonuclear leukocytes after activation of myeloperoxidase (inflammatory diseases). A possible mechanism of antioxidant and pro-oxidant activities of ß-carotene and carotenoid cleavage products is presented schematically in Fig. 3 . After moderate oxidative stress (left side), ß-carotene exerts antioxidant activity and macromolecules are protected against oxidation. Cleavage and oxidative products of carotenoids are accumulated under heavy oxidative stress (right side). Most are aldehydic in nature. The CCP-induced depletion of total protein-SH and GSH in mitochondria may be caused by direct reactions of aldehydes with sulfhydryl groups. Aldehydes may rapidly react with lysyl and histidine residues at low cellular levels. Consequently, mitochondrial proteins can be affected functionally. In fact, the decrease in SH-groups under the influence of several CCP was paralleled by inhibition of state 3 respiration. Based on our data, we identified the adenine nucleotide translocator as the part of oxidative phosphorylation that was functionally impaired. Interruption of the electron flow through the respiratory chain downstream of complex III leads to enhanced formation of reactive oxygen intermediates (O₂, H₂O₂). The increase in MDA levels, which reflects lipid peroxidation within mitochondria, indicated this increase in oxidative stress. Elevated accumulation of reactive oxygen species may induce oxidative damage to proteins, including the adenine nucleotide translocator, lipids, and DNA molecules in mitochondria, and the nucleus. Mitochondrial DNA exerts a pronounced susceptibility to oxidative stress because of the absence of histones and a lower capacity of DNA repair than the nucleus. Oxidative DNA damage increases the risk of cancer development. Our data may indicate a basic mechanism of the harmful effects of carotenoids in situations of heavy oxidative stress.

View larger version (38K): [in this window] [in a new window]   Figure 3. Hypothetic scheme concerning the mechanism of pro-oxidant and antioxidant effects of ß-carotene and carotenoid cleavage products. Under conditions of moderate oxidative stress (left side) the antioxidant effects of ß-carotene predominate. Under heavy oxidative stress (right side), the bulk of supplemented ß-carotene is oxidatively degraded leading to the formation of high amounts of cleavage products with pro-oxidant activities. Under these conditions, the pro-oxidative actions of ß-carotene products overcome the antioxidant activity of ß-carotene resulting in harmful effects. These are the impairment of adenine nucleotide translocator, accompanied by an increase in oxidative stress, indicated by reduction of protein sulfhydryl content, decreasing glutathione levels and redox state, and elevated accumulation of malonic dialdehyde. The increased levels of reactive oxygen species after inhibition of energy metabolism, particularly superoxide anion radicals and hydrogen peroxide, disturb the surrounding macromolecules, including proteins and nucleic acids, thereby increasing the risk of cancer.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0765fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0765fje

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