Photo-oxidation of 6-thioguanine by UVA: the formation of addition products with low molecular weight thiol compounds.

The thiopurine, 6-thioguanine (6-TG) is present in the DNA of patients treated with the immunosuppressant and anticancer drugs azathioprine or mercaptopurine. The skin of these patients is selectively sensitive to UVA radiation-which comprises >90% of the UV light in incident sunlight-and they suffer high rates of skin cancer. UVA irradiation of DNA 6-TG produces DNA lesions that may contribute to the development of cancer. Antioxidants can protect 6-TG against UVA but 6-TG oxidation products may undergo further reactions. We characterize some of these reactions and show that addition products are formed between UVA-irradiated 6-TG and N-acetylcysteine and other low molecular weight thiol compounds including ß-mercaptoethanol, cysteine and the cysteine-containing tripeptide glutathione (GSH). GSH is also adducted to 6-TG-containing oligodeoxynucleotides in an oxygen- and UVA-dependent nucleophilic displacement reaction that involves an intermediate oxidized 6-TG, guanine sulfonate (G(SO3) ). These photochemical reactions of 6-TG, particularly the formation of a covalent oligodeoxynucleotide-GSH complex, suggest that crosslinking of proteins or low molecular weight thiol compounds to DNA may be a previously unrecognized hazard in sunlight-exposed cells of thiopurine-treated patients.

Guanine sulphinate is a major stable product of photochemical oxidation of DNA 6-thioguanine by UVA irradiation.

The DNA of patients taking the immunosuppressant and anticancer drugs azathioprine or 6-mercaptopurine contains 6-thioguanine (6-TG). The skin of these patients is selectively sensitive to ultraviolet A radiation (UVA) and they suffer an extremely high incidence of sunlight-induced skin cancer with long-term treatment. DNA 6-TG interacts with UVA to generate reactive oxygen species, which oxidize 6-TG to guanine sulphonate (G(SO3)). We suggested that G(SO3) is formed via the reactive electrophilic intermediates, guanine sulphenate (G(SO)) and guanine sulphinate (G(SO2)). Here, G(SO2) is identified as a significant and stable UVA photoproduct of free 6-TG, its 2'-deoxyribonucleoside, and DNA 6-TG. Mild chemical oxidation converts 6-TG into G(SO2), which can be further oxidized to G(SO3)-a stable product that resists further reaction. In contrast, G(SO2) is converted back to 6-TG under mild conditions. This suggests that cellular antioxidant defences might counteract the UVA-mediated photooxidation of DNA 6-TG at this intermediate step and ameliorate its biological effects. In agreement with this possibility, the antioxidant ascorbate protected DNA 6-TG against UVA oxidation and prevented the formation of G(SO3).

Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions.

Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Photoactivation of DNA thiobases as a potential novel therapeutic option.

The thiopurines, 6-thioguanine and 6-mercaptopurine, are antileukemic agents that are incorporated into DNA following retrieval by the purine salvage pathway (see [1] for a review). Their toxicity requires active DNA mismatch repair (MMR), and thiopurine resistance is an acknowledged phenotype of MMR-defective cells [2, 3]. In addition to these direct cytotoxic effects, DNA thiobases have distinctive photochemical properties [4], the therapeutic potential of which has not been extensively evaluated. We report here that the thiopyrimidine nucleoside 4-thiothymidine is incorporated into DNA. It does not induce MMR-related toxicity, but it interacts synergistically with UVA light and dramatically sensitizes cultured human cells to very low, nonlethal UVA doses. 4-thiothymidine induced UVA dose enhancements of around 100-fold in DNA repair-proficient cells. Nucleotide excision repair-defective xeroderma pigmentosum cells were sensitized up to 1000-fold, implicating bulky DNA photoproducts in the lethal effect. The synergistic action of thiothymidine plus UVA required thymidine kinase, indicating a selective toxicity toward rapidly proliferating cells. Cooperative UVA cytotoxicity is a general property of DNA thiobases, and 6-thioguanine and 4-thiodeoxyuridine were also UVA sensitizers. Thiobase/UVA treatment may offer a novel therapeutic approach for the clinical management of nonmalignant conditions like psoriasis or for superficial tumors that are accessible to phototherapy.

An EPR study of the transfer and trapping of holes produced by radiation in guanine(thioguanine) hydrochloride single crystals.

Single crystals of guanine hydrochloride monohydrate, guanine hydrochloride dihydrate and anhydrous guanine dihydrochloride, doped with thioguanine, were irradiated with X and gamma rays. In all three systems the dominant radicals were associated with thioguanine. In the former two systems the stabilized species is the thiyl radical, formed by initial loss of an electron at some of the guanines in the crystal lattice, followed by hole migration to thioguanine and subsequent deprotonation of the radical formed. In the anhydrous guanine(thioguanine) dihydrochloride, that process is followed by acquisition of a chlorine ion. In the guanine hydrochloride monohydrate and guanine hydrochloride dihydrate lattices, systems of interacting closely spaced stacked bases and strings of chloride ions might support the migration of electrons and/or holes. In anhydrous guanine dihydrochloride, neither the bases nor the Cl- ions alone are capable of providing the means for the long-range electron, energy and spin transfer. It is the interchangeable sequence of the charged bases and the Cl- ions that makes the supporting strings or networks. The ultimate chlorination of the thioguanine-centered electron-loss radicals depends mainly on the availability of the Cl- ions and the space for their accommodation in the vicinity of the sulfur atom.