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Photocytotoxicity
and Photooxidative Damage to Cellular Nucleic Acids Sensitized by Nanomolar
Levels of Hypericin
Wamer, Wayne1
Food and Drug Administration1
Abstract-
Hypericin (HYP) is a major photoactive component in St. John s
Wort, a plant with rapidly increasing commercial importance. In addition
to their use as dietary supplements, extracts of St. Johns
Wort have recently been included in several cosmetic products marketed
for use on sun-exposed skin. This study was conducted to investigate
the photosensitizing potency of HYP. Two methods were used to assess
photosensitizing potency. First, to measure photocytotoxicity, human
skin fibroblasts were exposed for 18 hr to media supplemented with 80
nanomolar (nM) HYP. Fibroblasts were then irradiated through a layer
of phosphate-buffered saline with visible light (0 - 4.5 J/cm2).
Photocytotoxicity was then assessed as inhibition of colony formation.
Next, oxidation of cellular RNA and DNA photosensitized by HYP was examined.
Human skin fibroblasts were treated with the levels of HYP and visible
light described above. Immediately after irradiation, RNA and DNA were
isolated, enzymatically hydrolyzed to nucleosides and analyzed by HPLC.
Unmodified nucleosides were quantified with a UV detector. Products
of guanine oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)
in DNA and 8-oxo-7,8-dihydroguanosine (8-oxoG) in RNA, were quantified
with an electrochemical detector. We found that HYP was a potent sensitizer
of light-dependent cytotoxicity. Pre-incubation of fibroblasts with
80 nM HYP followed by irradiation with 2.2, 2.9, or 3.6 J/cm2
resulted in inhibition of colony formation by 20, 72 or 89%, respectively.
Similarly treated fibroblasts contained significant levels of oxidative
damage in RNA. Irradiation of HYP-treated fibroblasts with 0, 2.2, 2.9
or 3.6 J/cm2 resulted in 1.6 
0.2, 6.5
0.6, 12.3
0.5, or 15.0
1.1 8-oxoG/105G, respectively. No significant oxidative damage
in DNA was observed. These in vitro results suggest that very
low levels of exposure to HYP can result in phototoxicity and that photooxidative
damage plays a role in the phototoxicity of HYP.
Keywords: hypericin,
hydroxyguanine, oxidative, St. John's Wort
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