High-throughput metabolic toxicity screening using magnetic biocolloid reactors and LC-MS/MS

Abstract

An inexpensive, high-throughput genotoxicity screening method was developed by using magnetic particles coated with cytosol/microsome/DNA films as biocolloid reactors in a 96-well plate format coupled with liquid chromatography-mass spectrometry. Incorporation of both microsomal and cytosolic enzymes in the films provides a broad spectrum of metabolic enzymes representing a range of metabolic pathways for bioactivation of chemicals. Reactive metabolites generated via this process are trapped by covalently binding to DNA in the film. The DNA is then hydrolyzed and nucleobase adducts are collected using filters in the bottom for the 96-well plate of analysis by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). The magnetic particles facilitate simple and rapid sample preparation and workup. Major DNA adducts from ethylene dibromide, N-acetyl-2-aminofluorene and styrene were identified in proof-of-concept studies. Relative formation rates of DNA adducts correlated well with rodent genotoxicity metric TD(50) for the three compounds. This method has the potential for high-throughput genotoxicity screening, providing chemical structure information that is complementary to toxicity bioassays.

Figure 1

LC-MS SRM chromatograms and formation rate plot for styrene oxide DNA adducts using PDDA/DNA bioreactors with 5 mM styrene oxide. (A) βN⁷-SO-guanine with mass transition m/z 272>152 (t_R = 9.54 min), 5 min reaction; (B) αN³-SO-adenine with mass transition m/z 256>136 (t_R = 9.12 min), 5 min reaction. (C) Influence of reaction time on βN⁷-SO-guanine (■) and αN³-SO-adenine (), area ratio to internal standard 7-methylguanosine (m/z 298>166).

Figure 2

LC-MS/MS SRM chromatograms for deoxynucleosides: (A) deoxyadenosine, mass transition m/z 252>136 (t_R = 6.63 min), (B) deoxyguanosine, mass transition m/z 268>152 (t_R = 5.64 min), (C) deoxythymidine, mass transition m/z 243>127 (t_R = 5.79 min), (D) deoxycytidine, mass transition m/z 228>112 (t_R = 4.89 min).

Figure 3

LC-MS/MS analysis of reactions of magnetic biocolloid reactors with EDB (A, B and C) and AAF (D, E and F). (A) Representative SRM chromatogram with mass transition m/z 485>356 indicating the formation of S-[2-(N⁷-guanyl)ethyl]glutathione after 5 min reaction followed by neutral thermal hydrolysis. (B) Product ion spectrum of m/z 485 with inserted fragmentation. (C) Relative formation rate of S-[2-(N⁷-guanyl)ethyl]glutathione obtained from area ratio of analyte/internal standard. (D) Representative SRM chromatogram with mass transition m/z 447>331 indicating the formation of C8-AF-dGuo after 5 min reaction followed by enzyme hydrolysis. (E) Product ion spectrum of m/z 447 with inserted fragmentation. (F) Relative formation rate of C8-AF-dGuo from area ratio of analyte/internal standard.

Figure 4

Comparison of normalized DNA adduct formation rates obtained using magnetic biocolloid reactors and LC-MS/MS vs. the inverse of rodent carcinogenicity metric TD₅₀ (rat). (A) Overall formation rate of N⁷-SO-guanine and N³-SO-adenine from reaction with styrene; (B) S-[2-(N⁷-uanyl)ethyl]glutathione from enzyme reaction with EDB; (C) C8-AF-dGuo from enzyme reaction with AAF. The normalized formation rate was defined as, (peak area analyte/internal standard) min⁻¹ (mM of substrate)⁻¹.

Scheme 1

Experimental steps for metabolic toxicity screening using biocolloid reactors in a 96-well plate coupled with LC-MS/MS: (A) enzyme reactions are run; the center 96-well plate illustrates a possible multi-experiment design; (B) while particles are held in the wells by the magnetic plate, solution is replaced with a hydrolysis cocktail: (C) hydrolysis is done; (D) the magnet is moved to the top of the well plate to pull biocolloids away from the filters, and nucleobase/deoxynucleoside adduct samples are filtered into a second 96-well plate; and (E) samples in the second 96-well plate are analyzed by LC-MS/MS.

Scheme 2

Major metabolic pathways of styrene, ethylene dibromide and N-acetyl-2-aminofluorene leading to DNA adduct formation. (Adducts 3, 4 and 7 are presented in nucleobase form released by neutral thermal hydrolysis, and adduct 11 is presented in nucleoside form released by enzyme hydrolysis.)