Genotoxicity-Related Chemistry of Human Metabolites of Benzo[ghi]perylene (B[ghi]P) Investigated using Electro-Optical Arrays and DNA/Microsome Biocolloid Reactors with LC-MS/MS

Abstract

There is limited and sometimes contradictory information about the genotoxicity of polycyclic aromatic hydrocarbon benzo[ghi]perylene (B[ghi]P). Using recently developed metabolic toxicity screening arrays and a biocolloid reactor-LC-MS/MS approach, both featuring films of DNA and human metabolic enzymes, we demonstrated relatively low reactivity of metabolically activated B[ghi]P towards DNA. Electro-optical toxicity screening arrays showed that B[ghi]P metabolites damage DNA at a 3-fold lower rate than benzo[a]pyrene (B[a]P), whose metabolites have a strong and well-understood propensity for DNA damage. Metabolic studies using magnetic bead biocolloid reactors coated with microsomal enzymes in 96-well plates showed that cyt P450s 1A1 and 1B1 provide high activity for B[ghi]P and B[a]P conversion. Consistent with published results, the major metabolism of B[ghi]P involved oxidations at 3,4 and 11,12 positions, leading to formation of B[ghi]P 3,4-oxide and B[ghi]P 3,4,11,12-bisoxide. B[ghi]P 3,4-oxide was synthesized and reacted with deoxyadenosine at N6 and N7 positions and with deoxyguanosine at the N2 position. B[ghi]P 3,4-oxide is hydrolytically unstable and transforms into the 3,4-diol or converts to 3- or 4-hydroxy B[ghi]P. LC-MS/MS of reaction products from the magnetic biocolloid reactor particles coated with DNA and human enzymes revealed for the first time that a major DNA adduct results from reaction between B[ghi]P 3,4,11,12-bisoxide and deoxyguanosine. Results also demonstrated 5-fold lower formation rates of the major DNA adduct for B[ghi]P metabolites compared to B[a]P. Overall, results from both ECL array and biocolloid reactor-LC-MS/MS consistently suggest a lower human genotoxicity profile of B[ghi]P than B[a]P.

High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence

“…The characteristic fragmentation patterns of mass transition from m/z 570 to 454 for dG-BPDE, and that of m/z 554 to 257 for dA-BPDE confirm the generation of BPDE-DNA adducts. These results are consistent with our previous reports 43 and published literature. 44 …”

Chemical selectivity of nucleobase adduction relative to in vivo mutation sites on exon 7 fragment of p53 tumor suppressor gene

“…Our results are consistent with the fact that most damaged codons in tumors and cancer cell cultures involve guanines as the major site of attack by BPDE. 49 , 50 , 61 Guanines were also exclusive targets in the experiments with ss-oligonucleotides ( Table 2 ). Structural differences of ss-DNA and ds-DNA may play a role in sequence specific DNA damage.…”

Benzo[ghi]perylene Induces Morphological and Genotoxic Effects in Human Bronchial Cells Independently of The AhR Pathway

“…The increase of RNAm of the CYP1A1 and AHR genes is a classic marker of the AHR pathway activation that re ects the activation of the metabolism of phase I xenobiotics (Pan et al 2013). The present study, however, did not detect any increase in the transcription of the AHR and CYP1A1 genes at 6h of exposure of the NL-20 cells to B[ghi]p. This indicates that longer exposure times to B[ghi]p are required to activate the pathway, as was demonstrated at 24h of exposure, with the resulting transcriptional increase of AHR and CYP1A1.…”