Bracken Fern Carcinogen, Ptaquiloside, Forms a Guanine O6-Adduct in DNA

Abstract

Bracken fern (Pteridium sp.) is a viable and vigorous plant with invasive potential, ingestion of which causes chronic illness and cancers in farm animals. Bracken is a suspected human carcinogen, and exposure can result from ingestion of bracken-contaminated water, dairy products, or meat derived from livestock grazing on bracken fern. Bracken is also consumed in the diets of some communities. Ptaquiloside (PTQ), a known bracken carcinogen, is an illudane-type glycoside that forms a highly reactive electrophile, PTQ dienone, known to produce N⁷-guanine and N³-adenine adducts in DNA. Here, we demonstrate for the first time that PTQ dienone also produces an O⁶-alkylguanine (O⁶-PTBguanine) in DNA. Since O⁶-alkylguanines in DNA can be mutagenic, this work provides a potential mechanistic link between PTQ exposure and carcinogenicity. O⁶-PTBguanine is poorly repaired by O⁶-methylguanine-DNA methyltransferase that acts on other O⁶-alkylguanines, further highlighting the potential risk of exposure to bracken and PTQ.

Keywords: MGMT; O6-alkylguanine; bracken fern; carcinogenicity; ptaquiloside.

Scheme 1. Decomposition of Ptaquiloside to Ptaquilosin, Pterosin B, and PTQ Dienone That Can Cause DNA Damage

Figure 1

Structures of alkylated adenine and guanine formed following the reaction of DNA or guanosine with PTQ dienone: 1 and 2 have been identified in reactions with DNA following thermal hydrolysis/depurination and 2 and 3 have been identified in the reaction of PTQ dienone with guanosine.

Figure 2

ODNs used and characterization of O⁶-PTBG-containing ODN: (A) RP-HPLC chromatogram of purified ODN-1, 12.5–25% MeCN in 0.1 M TEAA pH 7.0 over 30 min; (B) 20% denaturing PAGE stained with Sybr Gold; (C) ESI MS of ODN-1; theoretical mass = 7201.627 Da, experimental mass = 7201.302 Da; and (D) nucleoside composition analysis of ODN-1 5′-GAACT(O⁶-PTBG]CAGCTCCGTGCTGGCCC) (see Table S1 in the Supporting Information).

Figure 3

Nucleoside composition analysis of the PTQ dienone-treated DNA duplex: (A) RP-HPLC following enzymatic digestion of PTQ dienone-treated DNA (see the Supporting Information for details); (B) extracted ion chromatogram (XIC) of the PTQ dienone-treated DNA duplex following enzymatic digestion to nucleosides and XIC of N⁷-PTB-dG/O⁶-PTB-dG 468.2 [M + H]⁺ (m/z); (C) MS/MS spectrum of selected ion 468.2 [M + H]⁺ (m/z), LC retention time 18.18 min; (D) MS/MS spectrum of selected ion 468.2 [M + H]⁺ (m/z), LC retention time 29.52 min; (E) RP-HPLC analysis of the O⁶-PTB-dG standard; (F) XIC of the 468.2 [M + H]⁺ (m/z) O⁶-PTB-dG standard shown in panel E; (G) MS of the O⁶-PTB-dG standard; and (H) MS/MS spectrum O⁶-PTB-dG. This corresponds to that shown in panel D, confirming the identity of O⁶-PTB-dG from the nucleoside digest. In panels C, D, and H, the masses of 352.17 and 201.127 m/z values are obtained from expected fragmentation across the glycosidic bond and pterosin B moiety, respectively, as shown.

Scheme 2. Synthesis of the O⁶-PTBdG Nucleoside Standard (5)

Figure 4

MGMT shows lack of repair of O⁶-PTBG: (A) PstI recognition sequence containing O⁶-alkG in the ODN duplex; (B) MGMT repair (alkyl transfer) results in an unmodified sequence that is cleaved by PstI; (C) PstI does not hydrolyze duplex DNA containing O⁶-alkG in its recognition sequence; and (D) analysis of ODN duplexes by 20% PAGE following treatment with MGMT and PstI: G represents unmodified duplex, PTBG indicates the ODN duplex containing O⁶-PTBG, Me indicates the ODN duplex containing O⁶-MeG.

Figure 5

EMSA of ds 23mer ODNs following incubation with increasing concentrations of MBP-Atl1. Duplexes were of unmodified guanine (G; ODNs 2 + 5 lanes 1–5), O⁶-PTBG (ODNs 1 + 5 lanes 7–11), and O⁶-MeG (ODNs 5 + 6). Lanes 13–17. The migration positions of the excess complement, unbound duplex, and bound duplex are shown.