Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.). 2. Palmatine, hydrastine, canadine, and hydrastinine.

Goldenseal is an herb that is widely used in dietary supplements, eye washes, and skin lotions. The presence of Goldenseal root powder in dietary supplements and the topical application of Goldenseal preparations raise the possibility that an adverse phototoxic reaction may result from an interaction between its constituent alkaloids and light in exposed tissues. We have previously shown that berberine, the major alkaloid in Goldenseal powder, in combination with UVA causes DNA damage and cell death in HaCaT keratinocytes [(2001) Chem. Res. Toxicol. 14, 1529]. We have studied the photochemical and photobiological properties of four minor alkaloids found in Goldenseal, namely, hydrastine, palmatine, canadine, and hydrastinine. UVA radiation of palmatine in aqueous solutions generated no (1)O(2), but in CH(2)Cl(2), copious amounts of (1)O(2) were detected (Phi = 0.2). Palmatine also photogenerated oxygen-centered radicals, (*)OH and O(2)(*)(-) in aerated aqueous buffer and acetonitrile, respectively, as detected by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In nitrogen-sparged acetonitrile containing DMPO, we observed the neutral palmatine radical formed by one-electron reduction. UVA irradiation (4 J/cm(2)) of HaCaT keratinocytes in the presence of palmatine (50 microM) resulted in a 50% decrease in cell viability but no DNA damage as measured by the comet assay. UVA irradiation of hydrastine, hydrastinine, or canadine (50 microM) did not cause DNA damage or cell death in keratinocytes. Although palmatine is photoactive, it is present in such small amounts in Goldenseal root powder that the phototoxicity of the herb is most likely due to berberine, the major constituent alkaloid.

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 1. Berberine.

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.

Experimental ruthenium plaque therapy of amelanotic and melanotic melanomas in the hamster eye.

The effects of beta-radiation on melanoma implanted into the hamster's eye were investigated. Two Bomirski hamster melanomas (BHMs), differing in their melanin content, were compared with regard to their radiosensitivity to ruthenium-106 (106Ru) radiation. Tumours growing in the iris were irradiated with 3, 6 or 10 Gy of 106Ru given as a single dose or in four fractions at 24 h Intervals. Tumour growth kinetics and distant metastases were studied, and the eyeballs were examined histologically. Dose-dependent delay of tumour growth was observed in both melanomas. After treatment with a dose of 6 Gy, the Ab amelanotic tumours grew 2.6 times slower, and the Ma melanotic tumours 1.4 times slower than untreated ones. The location of metastases differed in the two tested lines--pigmented metastases were found mainly in the lungs, while unpigmented metastases were found mainly in the kidneys. Histopathological analysis showed signs of blood vessel damage such as endothelial cells swelling, erythrocyte extravasation and tumour necrosis. This last finding increased with the rising dose of beta-radiation. Pigmented tumours were found to be two times more resistant to beta-radiation than amelanotic ones. The pattern of metastases of BHMs is determined by the type of melanoma (Ab or Ma). Exposure to beta-radiation from 106Ru did not significantly affect either the number or size of metastases except at a dose of 10 Gy. This dose caused a statistically significant decrease in the number of metastases in the Ma melanotic subline.

Gamma radiation and MC540 photosensitization of melanoma in the hamster's eye.

The purpose of this study was to evaluate the effects of cobalt-60 gamma-radiation and argon laser irradiation using injected merocyanine (MC540) as a photosensitizer on pigmented and non-pigmented Bomirski hamster melanomas growing in the eye. The animals were treated with one of four regimens, receiving gamma-irradiation only, photosensitization only, a combination of gamma-irradiation and photosensitization, or a combined time-fractionated treatment. Tumours were exposed to laser light 24 h after injection, when the photosensitizing dye concentration was highest. The degree of tissue damage was evaluated by observation of the area for necrosis, interruption of blood circulation, and the shape and dissemination of the tumour cells. Additionally, tumour growth was monitored through the measurement of tumour volume and also calculated from histological cross sections on the assumption that the tumour morphology is hemi-ellipsoidal. A single treatment of tumours by a combination of photodynamic therapy and ionizing radiation resulted in an additive effect, inhibiting tumour growth for 2-4 days. A time-fractionated treatment, given four times every 24 h, markedly delayed tumour growth for up to 6 weeks. The results indicate that MC540-mediated photodynamic treatment in combination with gamma-radiation exerts a significant therapeutic effect on a rapidly growing melanoma.

Photoproduction and direct spectral detection of singlet molecular oxygen (1O2) in keratinocytes stained with rose bengal.

In vivo, keratinocyte skin cells are exposed to photooxidative processes, some of which can be mediated by singlet molecular oxygen (1O2), a species that is very difficult to detect spectrally in cells. We photosensitized 1O2 in cultured HaCaT keratinocytes stained with rose bengal (RB) that localizes exclusively inside the keratinocyte hydrophobic regions, as evidenced by strongly red-shifted absorbance and intense fluorescence. We used keratinocytes grown in a monolayer on a plastic coverslip and in suspension. The phosphorescence spectrum (1200-1350 nm) from 1O2 was strongest when the coverslip containing RB-stained keratinocytes was irradiated in air. The spectral intensity decreased when the coverslip was immersed in D2O during irradiation and was almost completely quenched when it was irradiated while immersed in water. Water not only shortens the 1O2 lifetime but also reabsorbs part of the 1O2 phosphorescence, processes that do not occur when 1O2 is produced in a keratinocyte layer exposed to air. Because the RB was inside keratinocytes, singlet oxygen must also be produced inside the keratinocytes. However, the sensitivity to the extracellular environment suggests that most of the detectable 1O2 phosphorescence originates from those 1O2 molecules that escaped from the cell through its membrane into D2O or into the air, where 1O2 has longer lifetimes. Our results confirm directly that 1O2 is indeed photosensitized in living cells by RB. They also suggest that keratinocyte monolayers may be a good cell model to examine in vitro the production of 1O2 by other photosensitizers of environmental and photomedical interest.

[New technique for implanting Bomirski melanoma into the anterior chamber of Syrian hamster eyes]. / Nowa technika implantacji czerniaka Bomirskiego do przedniej komory oka chomika syryjskiego.

PURPOSE: To describe a newly elaborated ocular tumor model, obtained by implanting Bomirski melanoma into the anterior chamber of the eye in Syrian hamsters (Mesocricetus auratus). MATERIAL AND METHODS: The experiments were carried out using pigmented and non-pigmented lines of the tumor. Attempted injections of suspension of malignant melanoma cells in physiological salt solution to the anterior chamber with an infusion pump did not give the expected results. The growth of the tumor occurred only in 8% of eyes. The research material contained 132 adult pigmented hamsters. Tumor fragments of the size of 0,4-1,0 mm were implanted to the anterior chamber of the eyes. Melanotic cells were implanted in 71 hamsters, amelanotic ones in 61 hamsters. Hamsters were anesthetized with intravisceral vetbutal. The examination was carried out in 24 hour intervals. RESULTS: During the first 2 to 3 days the disappearance of the cells were observed, and then iris tumors appeared, in the cases of amelanotic cells after 4-6 days, and in the cases of melanotic cells after 8-10 days. Enucleated eye-balls were examined histopathologically. In all cases confirmation of the growth of melanoma in the iris was obtained.