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KAR‐2 (3′′‐(β‐chloroethyl)‐2′′,4′′‐dioxo‐3,5′′‐spiro‐oxazolidino‐4‐deacetoxy‐vinblastine) is a semisynthetic bis‐indol derivative, with high anti‐microtubular and anti‐tumour activities but with low toxicity. KAR‐2, in contrast to other biologically active bis‐indols (e.g. vinblastine), did not show anti‐calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR‐2 to calmodulin. The binding affinity of KAR‐2 to calmodulin (dissociation constant was about 5 μM) in the presence of Ca2+ was comparable to that of vinblastine. KAR‐2 was able to interact with apo‐calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR‐2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady‐state level. Comparison of the data obtained with KAR‐2 and bis‐indols used in chemotherapy suggests that the lack of anti‐calmodulin potency resides on the spiro‐oxazolidino portion of KAR‐2. This character of KAR‐2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti‐tumour test.

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Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.

Carotenoids are widely used as important micronutrients in food. Furthermore, carotenoid supplementation has been used in the treatment of diseases associated with oxidative stress such as various types of cancer, inflammatory diseases or cystic fibrosis. However, in some clinical studies harmful effects have been observed, e.g. a higher incidence of lung cancer in individuals exposed to extraordinary oxidative stress. The causal mechanisms of harmful effects are still unclear. Carotenoid breakdown products (CBPs) including highly reactive aldehydes and epoxides are formed during oxidative attacks in the course of antioxidative action. We investigated the formation of CBPs by stimulated neutrophils (and at further conditions), tested the hypothesis that CBPs may exert mitochondriotoxicity and tried to prevent toxicity in the presence of members of the antioxidative network. Stimulated neutrophils are able to degrade beta-carotene and to generate a number of CBPs. Concerning mitochondriotoxicity, we found that CBPs strongly inhibit state 3 respiration of rat liver mitochondria at concentrations between 0.5 and 20 microM. This was true for retinal, beta-ionone, and for mixtures of cleavage/breakdown products. The inhibition of mitochondrial respiration was accompanied by a reduction in protein sulfhydryl content, decreasing GSH levels and redox state, and elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favor functional deterioration in the adenine nucleotide translocator as a sensitive target. The presence of additional antioxidants such as alpha-tocopherol, ascorbic acid, N-acetyl-cysteine or others could mitigate mitochondriotoxicity. The findings reflect a basic mechanism of increasing the risk of cancer induced by carotenoid degradation products.