Francis P. Gasparro

WAVELENGTH DEPENDENCE FOR AMT CROSSLINKING OF pBR322 DNA

Abstract The wavelength dependence for 4′aminomethyl‐4,5′,8‐trimethylpsoralen crosslinking of a linearized plasmid DNA (pBR322) by narrow band UV‐A light (298–382 nm) has been determined. Maximal levels of crosslinking occurred with light in the 322–346 nm range. Crosslinks were shown to be photoreversible by shorter wavelength photons (298 and 310 nm). The correlation between the wavelength dependence for crosslink formation and the optimal wavelength for most psoralen action spectra further supports the notion that crosslinks are the major lesion responsible for the effectiveness of psoralen plus ultraviolet A therapies.

Inhibitory effect of 8-methoxypsoralen plus ultraviolet-A on interleukin-1 production by murine keratinocytes

We report the effects of 8‐methoxypsoralen (8‐MOP) plus ultraviolet‐A (UV‐A) irradiation on interleukin‐1 (IL‐1) production by murine epidermal keratinocytes, correlating its effect on IL‐1 with cell viability, DNA synthesis, and 8‐MOP‐DNA photoadduct formation. Freshly isolated murine keratinocytes were treated with various doses of 8‐MOP (5–100 ng/mL; incubation time, 30 min) plus 1 J/cm2 UV‐A and cultured for 1–3 days. The IL‐1/epidermal cell‐derived thymocyte‐activating factor (ETAF) activity in both supernatant and cell extract was reduced proportionately with increasing doses of 8‐MOP/UV‐A. Interleukin‐1 inhibitors induced by 8‐MOP plus UV‐A were not detected in either supernatant or cell extract. A clear reduction of the IL‐1 production was induced by the treatment as low as 15 ng/mL 8‐MOP plus 1 J/cm2 UV‐A, which led to the formation of 0.52 8‐MOP photoadducts per million DNA bases and affected neither cell viability nor DNA synthesis of the treated cells. Cells treated with 100 ng/mL 8‐MOP and 1 J/cm2 UV‐A exhibited 57% suppression of IL‐1 production in both 2‐ and 3‐day culture samples. This treatment resulted in the formation of 3.8 photoadducts per million bases as well as significant abrogation of DNA synthesis although cell viability was unchanged. These observations provide some insights into the phototoxicity mechanisms of 8‐MOP and the effect of PUVA therapy on the cytokine regulation in keratinocytes.

INVESTIGATION OF PROTRIPTYLINE PHOTOPRODUCTS WHICH CAUSE CELL MEMBRANE DISRUPTION

Abstract— Protriptyline (PTL; N‐methyl‐5H‐dibenzo[a,d]cycloheptene‐5‐propylamine) hydrochloride is a skin photosensitizing agent in humans. The fluorescence and photochemical behavior of PTL varies with the solvent. In water, 40% ethanol and ethanol in the hydrochloride salt of PTL has a fluorescence quantum yield of 0.81. The fluorescence quantum yield of PTL free base in selected organic solvents is between 0.41 and 0.17; in ethanol it remains at 0.81. Photolysis of PTL in acidic aqueous solution yields at least five photoproducts which were separated by high‐performance liquid chromatography. Three of these photoproducts lysed red blood cells. One of the photoproducts has been identified as a cyclobutyl photodimer of PTL based on its mass spectrum and UV absorption and its ability to undergo photore‐versal with 254 nm irradiation. The others were not cyclobutyl dimers. The yield of lytic products decreased as the ethanol content was increased and were not formed from PTL free base in any solvent.

HPLC analysis of 4',5'-monoadduct formation in calf thymus DNA and synthetic polynucleotides treated with UVA and 8-methoxypsoralen.

Abstract— 8‐methoxypsoralen monoadduct formation in calf thymus DNA irradiated with subbands of ultraviolet A light has been quantitated by HPLC analysis of the enzymatic hydrolysates of the DNA. Normalization of the yield of monoadducts for the variation in source output and the absorptivity of 8‐MOP at each of the irradiating wavelengths showed that the 4′,5′‐furan monoadduct was the principal photoproduct and the efficiency of its formation was independent of irradiating wavelength. Synthetic polynucleotides irradiated with ultraviolet A light demonstrated a base composition and sequence dependence for 8‐MOP photoreactivity: (poly(dAdT.dAdT) > poly(dA.dT) > poly(dGdC.dGdC) in both the B and Z forms > pofy(dT).

UVA and UVB‐Induced 8‐Methoxypsoralen Photoadducts and a Novel Method for their Detection by Surface‐Enhanced Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (SELDI‐TOF MS)

UVA‐activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320–400 nm) is more commonly used clinically, studies have shown that UVB (280–320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB‐induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm−1 M−1 at 300 nm) compared with the UVA region (2016 cm−1 M−1 at 365 nm), a greater extent of adduct formation is expected. SELDI‐TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A–T oligonucleotide. 8‐Methoxypsoralen (8‐MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI‐TOF. For UVB‐activated 8‐MOP, the extent of adducts was three times greater than for UVA. HPLC ESI‐MS analysis showed that UVB irradiation yielded high levels of 3,4‐monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4′,5′‐monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.