Fritz Böhm

β-Carotene with vitamins E and C offers synergistic cell protection against NOx

The peroxynitrite anion and the nitrogen dioxide (radical) are important toxic species which can arise in vivo from nitric oxide. Both in vivo and in vitro cell protection is demonstrated for β‐carotene in the presence of vitamin E and vitamin C. A synergistic protection is observed compared to the individual anti‐oxidants and this is explained in terms of an electron transfer reaction in which the β‐carotene radical is repaired by vitamin C.

Dietary carotenoids protect human cells from damage

A physical chemistry technique based on singlet oxygen luminescence at about 1270 nm and a biological cell membrane technique were used to study the quenching of singlet oxygen by four carotenoids bound to the surface of lymphoid cells. All the carotenoids studied showed a beneficial effect in cell protection, but there were subtle differences between them.

Dietary uptake of lycopene protects human cells from singlet oxygen and nitrogen dioxide - ROS components from cigarette smoke.

There is current interest in the health benefits of dietary carotenoids and the possible deleterious effects on certain sub-populations such as smokers. Here we report in vivo protection of human lymphocytes, conferred by dietary supplementation of lycopene rich foods against the reactive oxygen species, NO(2)(*) radical (by electron transfer) and 1(O)(2) (by energy transfer). It was found that a lycopene rich diet, maintained for 14 days, increased the serum lycopene level 10 fold compared to serum obtained after the same period, where a typical western European diet had been consumed. Relative lymphocyte protection factors of 17.6 and 6.3 against NO(2)(*) radical and 1(O)(2), respectively, were obtained, which re-enforce epidemiological data, showing protection against several chronic diseases by tomato lycopene.

ENHANCED PROTECTION OF HUMAN CELLS AGAINST ULTRAVIOLET LIGHT BY ANTIOXIDANT COMBINATIONS INVOLVING DIETARY CAROTENOIDS

Antioxidants like beta-carotene, alpha-tocopherol and ascorbic acid should be able to protect human cells against damage due to ultraviolet light. Cultured human fibroblasts have been irradiated with UVA or UVB light after incubation with the antioxidants or combinations of them. The efficiency of the protection by the antioxidants in dietary concentrations is estimated by cell counting following cell culture. In the case of UVA irradiation we find synergistic effects of combinations with beta-carotene as the main protector. On the other hand, only additive effects of the tested combinations are observed in the experiments with UVB light. Our experiments show a protective effect of dietary antioxidants against human tissue cell damage by ultraviolet light.

A UVA filter (4-tert-butyl-4′-methoxydibenzoylmethane): photoprotection reflects photophysical properties

Abstract A dibenzoylmethane-based UVA sunscreen, 4- tert -butyl-4′-methoxydibenzoylmethane has been shown to protect human lymphoid cells efficiently when present in high concentration (10 −2 M). The molecular mechanisms of this behaviour are proposed to be related to a mixture based on enol-transient-keto forms. At much lower sunscreen concentrations (10 −5 –10 −10 M) a switch from UVA to UVB protection is observed. This supports the theory of a conversion to a transient species absorbing in the UVB.

Antioxidant inhibition of porphyrin-induced cellular phototoxicity.

Porphyrins such as protoporphyrin IX (PP IX) and uroporphyrin I (UP I) can be phototoxic to human cells. To study the protective ability of antioxidants (beta-carotene, lycopene, ascorbic acid and alpha-tocopherol), against such porphyrin phototoxicity, membrane destruction experiments (Jurkat cells) and human cell cultures (fibroblasts) were performed. Both beta-carotene and lycopene and also the combination of beta-carotene, ascorbic acid and alpha-tocopherol offered cell protection against PP IX phototoxicity. Investigations of both cell membrane protection and of cell growth showed differences in terms of the protection afforded by the anti-oxidants. Thus, for PP IX, carotenoids alone, and in combination with ascorbic acid and alpha-tocopherol, showed higher protection factors in general than UP I. However, for membrane protection there was significant protection against UP I by the combination of beta-carotene, ascorbic acid and alpha-tocopherol but not by any of these anti-oxidants alone. The membrane protection against PP IX by beta-carotene, and especially lycopene, is significant presumably because of the high lipophilicity of all these molecules. However, the hydrophilic UP I will cause phototoxicity mainly via H(2)O(2), radical or singlet oxygen production in the aqueous phase, and these reactive species may be generated some distance from the cell membrane. This may lead to the little or no protection observed for UP I by the individual antioxidants. Nevertheless, a combination of beta-carotene, ascorbic acid and alpha-tocopherol offers membrane protection against the phototoxicity of both porphyrins. This is believed to occur as a result of synergistic processes. Our results suggest that the treatment of porphyria cutanea tarda and erythropoietic protoporphyria may be improved by the use of a combination of the antioxidants studied.

Different DNA repair time courses in human lymphoid cells after UVA, UVA1, UVB and PUVA in vitro.

The time course of DNA repair was investigated using the nucleoid sedimentation technique. Human lymphoid cells were irradiated with membrane related equitoxic doses of UVB, UVA/B, UVA and UVA1. Additionally, PUVA treatment of cells using two different light sources was performed. It was found that in the case of UVB and UVA/B irradiation of the cells, DNA repair activity arose lasting not longer than 4 h. However, in the case of UVA and UVA1 no repair activity was detectable with the used method but an increasing nucleoid density believed to indicate apoptosis. This could be shown with the same irradiation protocol. When the irradiation dose of UVA and UVA1 was lowered to one fifth of the equitoxic dose neither repair activity nor apoptosis was detected. There was no difference between UVA and UVA1 with respect to the nucleoid density time course. Hence, the initial absorbing chromophore is probably located in the UVA1 region. In the case of PUVA, we found DNA repair that was complete after 12 h. The UVB part in the irradiation spectrum of a PUVA system had an accelerating effect on the repair kinetics. The time course of DNA density appears to be related to subsequent cell behaviour: apoptosis or incomplete DNA repair including the risk of mutation.

BILIRUBIN PHOTOTOXICITY TO HUMAN CELLS BY GREEN LIGHT PHOTOTHERAPY in vitro

Phototherapy of newborn infants with blue or green light is the most common treatment of neonatal hyperbilirubinemia. Using bilirubin bound to human lymphoid and basal skin cells we obtained the green light dose dependency of the bilirubin phototoxicity to these cell types. Cells (3–5× 106/mL) were incubated with bilirubin complexed to human serum albumin (final concentrations 340 μM bilirubin, 150 μM albumin). Under these conditions all cells showed maximum binding of bilirubin. Irradiation with broadband green light (Λmax= 512 nm) over 24 h led to a light dose‐dependent population of cells, which contained no bilirubin on the cell membrane as determined by Nomarski interference microscopy. The light‐induced mechanism of the disappearance of bilirubin caused lethal membrane damage to the cells (trypan blue exclusion test). The cell kill rate increased with the irradiation dose and with the fraction of cells with no bilirubin. When 90% of lymphoid cells were bilirubin free, 46% of them were dead (using 480 J cm−1 green light). Similar results were obtained with basal skin cells. In addition, bilirubin‐induced damage of cell membrane and nuclear membrane was also shown by transmission electron microscopy. Bilirubin (340 μM) in the dark led to 5% of the cells being killed. Basal skin cells bind 2.5 times more bilirubin molecules than lymphoid cells and showed a different bilirubin disappearance. Irradiation of bilirubin in carbon tetrachloride with 514.5 nm laser light showed generation of singlet oxygen via its luminescence at 1270 nm. These results demonstrate that green light phototherapy of hyperbilirubinemia may cause both skin and immune system damage.

Fourier-transform detection of singlet oxygen and fluorescence from cell membrane bound porphyrins

A weak luminescence due to singlet oxygen O2(a 1Δg) and a stronger fluorescence emission from a porphyrin bound to the surface of living cells have been obtained using a Fourier-transform interferometric technique. The porphyrin fluorescence shows a marked wavelength shift compared with that observed when the cells are not present. The singlet oxygen emission is accompanied by cell membrane destruction as shown by the Trypan Blue exclusion test.