Goldenseal is an herb which is widely used for many medical applications such as in eyewashes and skin lotions and which is currently undergoing testing by the National Toxicology Program. The main alkaloid constituent of Goldenseal is berberine. The topical application of Goldenseal or berberine to the skin or eyes raises the possibility that an adverse phototoxic reaction may result from an interaction between the alkaloid and light. We have therefore studied the photochemistry of berberine in different solvents and its phototoxicty to HaCaT keratinocytes. Irradiation of berberine in aqueous solutions does not generate (1)O(2), but in CH(2)Cl(2), (1)O(2) is produced with a quantum yield phi = 0.34. With the aid of the electron paramagnetic resonance (EPR) spin trapping technique and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we have detected oxygen-centered radicals photogenerated by berberine in water and acetonitrile. In the latter solvent and in the absence of oxygen, the neutral berberine radical formed by one electron reduction was observed. Methanol radicals were detected by EPR in water/alcohol low-temperature glasses irradiated in the berberine long-wavelength absorption band. In such alcoholic glasses, we have also detected an EPR signal from the berberine triplet at 77 K, in contrast to aqueous glasses where neither triplet nor radicals were detectable. Our data show that, although a weak photosensitizer in water, berberine is able to produce both (1)O(2) and radical species in a nonpolar environment. UVA irradiation of HaCaT keratinocytes in the presence of 50 microM berberine resulted in an 80% decrease in cell viability and a 3-fold increase in DNA damage as measured by the Comet assay. These findings suggest that exposure to sunlight or artificial light sources emitting UVA should be avoided when topical preparations derived from Goldenseal or containing berberine are used.
The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.
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