Translesion synthesis of 6-nitrochrysene-derived 2ʹ-deoxyadenosine adduct in human cells
Graphical abstract
Introduction
Nitropolycyclic aromatic hydrocarbons (NO2-PAHs) are common environmental pollutants as they are formed during combustion of diesel and other fossil fuels [[1], [2], [3], [4]]. They are also present in certain foods and beverages. The International Agency for Research on Cancer (IARC) labeled diesel exhaust as “carcinogenic to humans” (Group 1) and one of its NO2-PAH contaminant 6-nitrochrysene (6-NC) as “probably carcinogenic to humans” (Group 2A) [5]. 6-NC is mutagenic in bacteria and carcinogenic in experimental animals [[6], [7], [8], [9]]. It is the most potent carcinogen ever tested in the newborn mouse assay and its carcinogenic potency in rat mammary gland is higher than that of the potent carcinogens benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [[9], [10], [11]].
6-NC is metabolically activated by two different pathways: the first involves nitroreduction to form 6-hydroxyaminochrysene (N−OH-6-AC) and the second involves both nitroreduction and ring oxidation. N−OH-6-AC, generated in the first pathway forms three DNA adducts, N-(dG-8-yl)-6-AC (1), 5-(dG-N2-yl)-6-AC (2), and N-(dI-8-yl)-6-AC (4), the latter being produced by deamination of the 2ʹ-deoxyadenosine adduct, N-(dA-8-yl)-6-AC (3) (Scheme 1) [[12], [13], [14], [15]]. The second pathway generates trans-1,2-dihydroxy-1,2-dihydro-6-hydroxyaminochrysene (1,2-DHD-6-NHOH-C), which forms 5-(dG-N2-yl)-1,2-DHD-6-AC, N-(dG-8-yl)-1,2-DHD-6-AC, and N-(dA-8-yl)1,2-DHD-6-AC [14]. All these DNA adducts have been detected in several organs of rats treated with 6-NC orally or by intraperitoneal injection [12,14].
When the nucleotide excision repair (NER) efficiencies of N-(dG-8-yl)-1,2-DHD-6-AC, N-(dG-8-yl)-6-AC, and N-(dA-8-yl)-6-AC were compared in HeLa cell extracts, N-(dG-8-yl)-6-AC repair was most facile [16], even though N-(dG-8-yl)-6-AC was estimated to be 15-fold more resistant to NER than the cis-Pt adduct [17]. NER of N-(dG-8-yl)-1,2-DHD-6-AC is ∼2-fold more resistant than N-(dG-8-yl)-6-AC whereas N-(dA-8-yl)-6-AC is repaired 8-fold more slowly than N-(dG-8-yl)-6-AC [16]. It was speculated that slow repair of the 6-NC adducts and thus their persistence in mammalian tissues play a part in the carcinogenicity of 6-NC [16]. However, mutagenicity of these adducts in mammalian cells have never been reported.
We have recently developed a total synthesis method to prepare site-specifically incorporated N-(dA-8-yl)-6-AC in any desired sequence [18]. We have also shown that this adduct is mutagenic in Escherichia coli. In this article, we report the mutagenicity of this adduct in human embryonic kidney (HEK) 293T cells. We also determined the roles of the translesion synthesis (TLS) DNA polymerases on the bypass and mutagenicity of the DNA adduct.
Section snippets
Materials
All materials, including reagents and solvents, were of commercial grade. [γ-32 P] ATP was purchased from Perkin Elmer Health Sciences Inc. (Shelton, CT). The enzymes were purchased from New England Biolabs (Beverly, MA). All unmodified oligodeoxynucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Synthetic siRNA duplex against hPol η (SI02663619), hPol κ (SI04930884), and negative control siRNA (1,027,280) were purchased from Qiagen (Valencia, CA). HEK 293 T cells with
Experimental system
Our experimental system involves construction of an N-(dA-8-yl)-6-AC containing single-stranded plasmid pMS2 that carries f1 and SV40 origins of replication, neomycin and ampicillin resistance genes, the SV40 early promoter, SV40 small tumor antigen splice sites, and SV40 early poly-adenylation site, which enable it to be replicated in both mammalian cells and E. coli [24]. The lesion containing pMS2 construct was mixed with an unmodified plasmid that contained a different DNA sequence at the
Conclusions
While mutagenicity of many bulky dG adducts in mammalian cells have been published [33], there is a paucity of reports on the mutagenicity of bulky dA adducts. Of the limited number of studies on the mutagenic outcome of bulky dA adducts [[34], [35], [36], [37], [38], [39], [40]], none has evaluated a bulky C8-dA adduct. To our knowledge, this is the first study on replication of a bulky NO2-PAH-derived C8-dA adduct in human cells, which showed that its bypass relies heavily on the TLS
CRediT authorship contribution statement
Brent V. Powell: Investigation, Writing - review & editing. Jan Henric T. Bacurio: Investigation. Ashis K. Basu: Conceptualization, Investigation, Funding acquisition, Supervision, Writing - original draft, Writing - review & editing.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the NIEHS (grant ES023350).
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