Biological activities of natural and synthetic carotenoids: induction of gap junctional communication and singlet oxygen quenching.

Induction of gap junctional communication (GJC) and antioxidant activities of carotenoids have been considered as biochemical mechanisms underlying the cancer-preventive properties of these compounds. beta-Carotene and other carotenoids, including those lacking provitamin A activity, proved to be active in both these parameters. The beta-carotene analogs retrodehydro-beta-carotene, echinenone, cryptoxanthin (3-hydroxy-beta-carotene), 4-hydroxy-beta-carotene and canthaxanthin stimulate GJC and efficiently deactivate singlet molecular oxygen. beta-Carotene is less active than its retro-dehydro analog with respect to (1)O2-quenching but GJC is similar. The five-membered ring analog of canthaxanthin, dinor-canthaxanthin, has less effect on GJC as compared with the parent compound but exhibits increased singlet oxygen quenching. Straight-chain polyene dialdehydes are quenchers of singlet oxygen, the efficiency increasing with the number of conjugated double bonds. However, none of these compounds significantly induce GJC. These data indicate that the two properties of carotenoids addressed in this study may operate independent of each other.

Induction of gap junctional communication by 4-oxoretinoic acid generated from its precursor canthaxanthin.

The activity of 4-oxoretinoic acid as an inducer of gap junctional communication was investigated in C3H/10T1/2 murine fibroblasts. Two isomers of this retinoid, all-trans- and 13-cis-4-oxoretinoic acid, enhance gap junctional communication. This is accompanied by increased expression of connexin43 mRNA. Decomposition fractions of canthaxanthin were isolated by preparative high-performance liquid chromatography and shown to be active in the cell-cell communication assay. Two of the decomposition compounds were identified as all-trans- and 13-cis-4-oxoretinoic acid. Therefore, it is concluded that the biological activity of canthaxanthin regarding cell-cell communication is at least in part due to the formation of active decomposition products such as 4-oxoretinoic acid.

Efficacy of all-trans-beta-carotene, canthaxanthin, and all-trans-, 9-cis-, and 4-oxoretinoic acids in inducing differentiation of an F9 embryonal carcinoma RAR beta-lacZ reporter cell line.

A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation.

Vitamin D influences gap junctional communication in C3H/10T 1/2 murine fibroblast cells.

Vitamin D3, cholecalciferol, induces cell-cell communication via gap junctions in murine fibroblasts (C3H/10T 1/2 cells) at concentrations between 0.01 and 1.0 microM, as assayed by the dye transfer method using Lucifer yellow CH. The extent of induction is similar to that obtained with the positive controls, canthaxanthin or retinoic acid, applied at 10 and 1 microM, respectively. Vitamin D2 also induces cell-cell communication. At elevated concentrations of vitamin D3 (5 microM) there is a suppression of gap junctional communication, reversible upon exposure to all-trans retinoic acid (1 microM) after removal of vitamin D3 from the medium. Conversely, communication between cells prestimulated with retinoic acid (1 microM) rapidly decreases when the retinoid is replaced by vitamin D3 (5 microM). The data demonstrate a role for vitamin D in the regulation of intercellular communication. This novel property of vitamin D may contribute to the antiproliferative effects of vitamin D exhibited in some types of cancer.

Separation of beta-carotene and lycopene geometrical isomers in biological samples.

The analysis of beta-carotene and lycopene, the two predominant carotenoids in human serum and tissues, was extended to the level of geometrical (cis-trans) isomers by using an improved reversed-phase HPLC methodology. We separated five geometrical isomers of beta-carotene and seven of lycopene in human serum and tissues. 13-cis-beta-Carotene was identified as the predominant cisisomer in human serum, contributing about 5% to total beta-carotene. In tissue, however, considerable amounts of 9-cis- and traces of 15-cis-beta-carotene were also detected. In contrast to beta-carotene, the lycopene isomer patterns in human serum and tissues are quite similar.

Cis/trans isomerization of carotenoids by the triplet carbonyl source 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane.

The interaction of biological carotenoids with 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), a thermodissociable source of electronically excited ketones, was investigated using reversed-phase high-performance liquid chromatography. Incubation of the all-trans isomers of beta-carotene, lycopene and canthaxanthin with HTMD led to significant trans-to-cis isomerization, with cis isomers accounting for 20-50% of products formed (the balance assigned as oxidation products). The isomers forming from all-trans-beta-carotene were identified as 9-cis-, 13-cis- and 15-cis-beta-carotene by cochromatography of cis isomer standards and by on-line diode array absorbance spectroscopy. An HTMD-dependent cis-to-trans isomerization was observed in incubations started with 15-cis-beta-carotene, and it occurred more rapidly and to a greater extent than the isomerization of all-trans-beta-carotene. The isomer patterns generated from lycopene and beta-carotene are generally similar to those reported recently for various human tissues (Stahl et al., 1992, Arch. Biochem. Biophys. 294, 173-177).