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. 2019 Dec;60(9):792-806.
doi: 10.1002/em.22321. Epub 2019 Aug 16.

Bioactivation mechanisms of N-hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

Yoshiharu Okuno  1   2 , Radha Bonala  3 , Sivaprasad Attaluri  3 , Francis Johnson  3   4 , Arthur P Grollman  3   5 , Viktoriya S Sidorenko  3 , Yoshimitsu Oda  6
Affiliations

Bioactivation mechanisms of N-hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids

Yoshiharu Okuno et al. Environ Mol Mutagen. 2019 Dec.

Abstract

Aristolochic acids (AAs) are human nephrotoxins and carcinogens found in concoctions of Aristolochia plants used in traditional medicinal practices worldwide. Genotoxicity of AAs is associated with the formation of active species catalyzed by metabolic enzymes, the full repertoire of which is unknown. Recently, we provided evidence that sulfonation is important for bioactivation of AAs. Here, we employ Salmonella typhimurium umu tester strains expressing human N-acetyltransferases (NATs) and sulfotransferases (SULTs), to study the role of conjugation reactions in the genotoxicities of N-hydroxyaristolactams (AL-I-NOH and AL-II-NOH), metabolites of AA-I and AA-II. Both N-hydroxyaristolactams show stronger genotoxic effects in umu strains expressing human NAT1 and NAT2, than in the parent strain. Additionally, AL-I-NOH displays increased genotoxicity in strains expressing human SULT1A1 and SULT1A2, whereas AL-II-NOH shows enhanced genotoxicity in SULT1A1/2 and SULT1A3 strains. 2,6-Dichloro-4-nitrophenol, SULTs inhibitor, reduced umuC gene expression induced by N-hydroxyaristolactams in SULT1A2 strain. N-hydroxyaristolactams are also mutagenic in parent strains, suggesting that an additional mechanism(s) may contribute to their genotoxicities. Accordingly, using putative SULT substrates and inhibitors, we found that cytosols obtained from human kidney HK-2 cells activate N-hydroxyaristolactams in aristolactam-DNA adducts with the limited involvement of SULTs. Removal of low-molecular-weight reactants in the 3.5-10 kDa range inhibits the formation of aristolactam-DNA by 500-fold, which could not be prevented by the addition of cofactors for SULTs and NATs. In conclusion, our results demonstrate that the genotoxicities of N-hydroxyaristolactams depend on the cell type and involve not only sulfonation but also N,O-acetyltransfer and an additional yet unknown mechanism(s). Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Keywords: N,O-acetyltransfer; umu test; HK-2 cytosols; aristolactam-DNA adducts; sulfonation.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Proposed pathways for the metabolic activation of the aristolochic acids. Four‐electron NR of AA‐I and AA‐II produces their respective N‐hydroxyaristolactams, AL‐I‐NOH and AL‐II‐NOH. N‐hydroxyaristolactams decompose to form electrophilic cyclic nitrenium/carbenium ions with delocalized positive charge, or undergo conjugation reactions catalyzed by sulfotransferases (SULTs) and N‐acetyltransferases (NATs). Resulting N‐sulfonyloxyaristolactams (AL‐I‐NOSO3H and AL‐II‐NOSO3H) and N‐acetoxylaristolactams (AL‐I‐NOAc and AL‐II‐NOAc) readily undergo solvolysis, forming the same active species as N‐hydroxyaristolactams. N‐hydroxyaristolactams are more stable than their esters and lead to DNA adduction at much slower rates.
Figure 2
Figure 2
Induction of umuC gene expression (A and B) and cytotoxicity response (C and D) induced by AL‐I‐NOH and AL‐II‐NOH in S. typhimurium tester strains TA1535/pSK1002 (◆), NM2009 (■) and NM2000 (●). Experiments were carried out as described in the section Material and Methods. In (A) and (B), each point was derived as the ratio of β‐galactosidase activity for each dose of the compound to the activity in the corresponding untreated cells. In all graphs, points represent mean values of triplicate determinations.
Figure 3
Figure 3
Induction of umuC gene expression (A and B) and cytotoxicity response (C and D) induced by AL‐I‐NOH and AL‐II‐NOH in S. typhimurium tester strains NM6000 (◆), NM6001 (■), and NM6002 (●). In (A) and (B), results are presented as values corresponding to β‐galactosidase activity normalized to untreated control. In all graphs, points are mean values of triplicate determinations.
Figure 4
Figure 4
Induction of umuC gene expression (A and B) and cytotoxicity response (C and D) induced by AL‐I‐NOH and AL‐II‐NOH in S. typhimurium tester strains NM7000 (◆), NM7001 (■), NM7002 (●), and NM7003 (▲). Experiments were conducted as described in the section Material and Methods. In (A) and (B), values for β‐galactosidase activity, observed for each dose of compound, were divided by that measured in corresponding untreated strain. In all graphs, points represent mean values of triplicate determinations.
Figure 5
Figure 5
Activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols, fortified, and not fortified by PAPS. Cytosols from HK‐2 cells (6; 25; 100; 400; 1500; 6250 and 25,000 ng/100 μL) or recombinant SULT1B1 protein (1 ng/100 μL) were incubated with 100 μM AL‐I‐NOH in the presence of ssDNA with or without 0.2 mM PAPS for 2 h at 37°C. DNA was extracted and two micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. (A), (B), and (D) are representative fragments of polyacrylamide gels following electrophoresis and exposure for 2 (A and B) or 10 min (D) on a phosphoscreen. (A) and (B) are unaltered fragments of the same gel shown at the same contrast, and analysis in (D) was done in a separate experiment and shown at the similar contrast level but longer exposure time. Control 1 in (A) is DNA/cytosol; Control 2—DNA/AA‐I; Controls 3–5—DNA/AL‐I‐NOH; Control 6—DNA/AL‐I‐NOH/PAPS. AL‐DNA levels in control incubations with AL‐I‐NOH were at 0.2 ± 0.02 adducts/106 nucleotides. (C) Quantitative representation of results shown in (B). Filled circles—reactions with PAPS; empty circles—without PAPS. (D) s1 (8 AL‐DNA/105 nucleotides) and s2 (6 AL‐DNA/105 nucleotides) represent adducted DNA from reactions conducted with PAPS from R&D and Sigma‐Aldrich, respectively. St—standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 30 fmol each. AL‐DNA is a combined term for dG‐AL and dA‐AL adducts.
Figure 6
Figure 6
Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 107 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 107 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.
Figure 7
Figure 7
Effect of PAP, quercetin, and b‐estradiol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 50 μM of AL‐I‐NOH in the presence of ssDNA for 30 min at 37°C. Reactions were set in triplicate for each condition, without or with 100 μM one of the following: PAP, quercetin and β‐estradiol. DNA was extracted and five micrograms from each sample was subjected to adduct analysis. Fragment of PAGE and quantification of obtained results are shown. St‐ mixture of standard at 30 fmol each. dG‐ and dA‐AL are upper and lower bands, respectively.
Figure 8
Figure 8
Effect of dialysis on 3.5 kDa MWCO and prolonged incubation at 4°C on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. Cytosols from HK‐2 cells were dialyzed against Tris–HCl buffer (pH 7.5) using 3.5 MWCO membrane overnight at 4°C. In parallel, a portion of cytosol was incubated above the membrane without the dialysis overnight at 4°C. 100–5000 ng of the protein was incubated with ssDNA and 100 μM AL‐I‐NOH with or without PAPS, as indicated. Reactions were allowed to run for 45 min at 37°C. DNA (5 μg) was used for adduct analysis. Fragments of PAGE (left panel) and quantification of obtained results (right panel) are shown. St‐ mixture of standard at 60 fmol each. Upper band is dG‐AL, and lower band is dA‐AL.
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