7-N-Acetylcysteine-pyrrole conjugate-A potent DNA reactive metabolite of pyrrolizidine alkaloids

Abstract

Plants containing pyrrolizidine alkaloids (PAs) are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs) that are capable of alkylating cellular DNA and proteins, form (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP) and DHP. Reactions of 7-NAC-DHP with 2'-deoxyguanosine (dG), 2'-deoxyadenosine (dA), and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation.

Keywords: 7-N-acetylcysteine-DHP; DHP; LC-ES-MS/MS; pyrrolizidine alkaloid.

Figure 1

Structures of PAs, dehydro-PAs, DHP, DHP-dG-1, DHP-dG-2, DHP-dG-3, DHP-dG-4, DHP-dA-1, DHP-dA-2, DHP-dA-3, and DHP-dA-4 adducts. dA = 2′-deoxyadenosine, dG = 2′-deoxyguanosine, DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, PA = pyrrolizidine alkaloids.

Figure 2

HPLC profiles of 7-NAC-DHP and DHP obtained from (A) organic synthesis; and from metabolism of riddelliine in the presence of N-acetylcysteine by (B) male human liver microsomes; (C) male rat liver microsomes; and (D) male rat liver microsomes without the addition of NADPH. DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, HPLC = high performance liquid chromatography, NAC = N-acetylcysteine.

Figure 3

Electrospray positive ion/product ion mass spectra of 7-NAC-DHP from (A) organic synthesis; and in the chromatographic peak eluting at 28.85–29.25 minutes in Fig. 2 obtained from the results of the metabolism of riddelliine in the presence of N-acetylcysteine by (B) male human liver microsomes; (C) male rat liver microsomes; and (D) male rat liver microsomes without the addition of NADPH. The product ions of the [M – H₂O + H]⁺ m/z 281 were acquired with a collision energy of 10 eV. DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, NAC = N-acetylcysteine.

Figure 4

Electrospray positive ion/product ion mass spectra of DHP from (A) organic synthesis; and in the chromatographic peak eluting at 25.3 minutes in Fig. 2 obtained from the metabolism of riddelliine in the presence of N-acetylcysteine by (B) male human liver microsomes; (C) male rat liver microsomes; and (D) male rat liver microsomes in the absence of NADPH, The product ions of the [M – H₂O + H]⁺ m/z 136 were acquired with a collision energy of 22 eV. DHP = (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine.

Figure 5

HPLC profiles of 7-NAC-DHP and DHP from (A) organic synthesis; and from metabolism of monocrotaline in the presence of N-acetylcysteine by (B) male human liver microsomes; and (C) male rat liver microsomes. DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, NAC = N-acetylcysteine.

Figure 6

Electrospray positive ion/product ion mass spectra of 7-NAC-DHP from (A) organic synthesis; and in the chromatographic peak eluting at 28.85–29.25 minutes in Fig. 5 obtained from the metabolism of monocrotaline in the presence of N-acetylcysteine by (B) male human liver microsomes; and (C) male rat liver microsomes. The product ions of the [M – H₂O + H]⁺ m/z 281 were acquired with a collision energy of 10 eV. DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, NAC = N-acetylcysteine.

Figure 7

Electrospray positive ion/product ion mass spectra of DHP from (A) organic synthesis; and in the chromatographic peak eluting at 25.3 minutes in Fig. 5 obtained from the metabolism of monocrotaline in the presence of N-acetylcysteine by (B) male human liver microsomes; and (C) male rat liver microsomes. The product ions of the [M – H₂O + H]⁺ m/z 136 were acquired with a collision energy of 22 eV. DHP = (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine.

Figure 8

HPLC profiles of reactions of 7-NAC-DHP with dG and dA for 1, 3, and 5 days: (A–C) detection of DHP-dG adducts monitored at 256 nm; and (D–F) detection of DHP-dA adducts monitored at 269 nm. For reaction conditions, see Materials and methods section. dA = 2′-deoxyadenosine, dG = 2′-deoxyguanosine, DHP = (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine.

Figure 9

The formation of DHP-dG and DHP-dA adducts from reaction of 7-NAC-DHP with calf thymus DNA for 1, 3, and 5 days. dA = 2′-deoxyadenosine, dG = 2′-deoxyguanosine, DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, NAC = N-acetylcysteine.

Figure 10

LC/MS SRM chromatograms of DHP-dG and DHP-dA adducts formed from the reaction of 7-NAC-DHP with calf thymus DNA for 3 days. dA = 2′-deoxyadenosine, dG = 2′-deoxyguanosine, DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, IS = DHP-[¹⁵N₅]dG and DHP-[¹⁵N₅, ¹³C₁₀]dA labeled internal standards, LC/MS = liquid chromatography/mass spectrometry, NAC =N-acetylcysteine, SRM = selected reaction monitoring.

Figure 11

The proposed general metabolic activation pathways leading to the formation of DNA adducts and the potential initiation of PA-induced liver tumors. dA = 2′-deoxyadenosine, dG = 2′-deoxyguanosine, DHP =(±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, NAC =N-acetylcysteine, PA = pyrrolizidine alkaloids.