48 research outputs found
Lack of recognition by global-genome nucleotide excision repair accounts for the high mutagenicity and persistence of aristolactam-DNA adducts
Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by Aā:āT to Tā:āA transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues
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SYNTHESIS OF THE FULLY PROTECTED PHOSPHORAMIDITE OF THE BENZENE-DNA ADDUCT, N2- (4-HYDROXYPHENYL)-2'-DEOXYGUANOSINE AND INCORPORATION OF THE LATER INTO DNA OLIGOMERS
N2-(4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2'-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers
DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells
Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ā¼50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dAāT and AL-II-dGāT transversions
Inactivation of PPARĪ²/Ī“ adversely affects satellite cells and reduces postnatal myogenesis.
Peroxisome proliferator-activated receptor Ī²/Ī“ (PPARĪ²/Ī“) is a ubiquitously expressed gene with higher levels observed in skeletal muscle. Recently, our laboratory showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that PPARĪ²/Ī“ modulates myostatin activity to induce myogenesis in skeletal muscle. In the present study, we show that PPARĪ²/Ī“-null mice display reduced body weight, skeletal muscle weight, and myofiber atrophy during postnatal development. In addition, a significant reduction in satellite cell number was observed in PPARĪ²/Ī“-null mice, suggesting a role for PPARĪ²/Ī“ in muscle regeneration. To investigate this, tibialis anterior muscles were injured with notexin, and muscle regeneration was monitored on days 3, 5, 7, and 28 postinjury. Immunohistochemical analysis revealed an increased inflammatory response and reduced myoblast proliferation in regenerating muscle from PPARĪ²/Ī“-null mice. Histological analysis confirmed that the regenerated muscle fibers of PPARĪ²/Ī“-null mice maintained an atrophy phenotype with reduced numbers of centrally placed nuclei. Even though satellite cell numbers were reduced before injury, satellite cell self-renewal was found to be unaffected in PPARĪ²/Ī“-null mice after regeneration. Previously, our laboratory had showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that inactivation of PPARĪ²/Ī“ increases myostatin signaling and inhibits myogenesis. Our results here indeed confirm that inactivation of myostatin signaling rescues the atrophy phenotype and improves muscle fiber cross-sectional area in both uninjured and regenerated tibialis anterior muscle from PPARĪ²/Ī“-null mice. Taken together, these data suggest that absence of PPARĪ²/Ī“ leads to loss of satellite cells, impaired skeletal muscle regeneration, and postnatal myogenesis. Furthermore, our results also demonstrate that functional antagonism of myostatin has utility in rescuing these effects
Total Synthesis of the Aristolochic Acids, Their Major Metabolites, and Related Compounds
Plants from the <i>Aristolochia</i> genus have been recommended
for the treatment of a variety of human ailments since the time of
Hippocrates. However, many species produce the highly toxic aristolochic
acids (AAs), which are both nephrotoxic and carcinogenic. For the
purposes of extensive biological studies, a versatile approach to
the synthesis of the AAs and their major metabolites was devised based
primarily on a SuzukiāMiyaura coupling reaction. The key to
success lies in the preparation of a common ring-A precursor, namely,
the tetrahydropyranyl ether of 2-nitromethyl-3-iodo-4,5-methylendioxybenzyl
alcohol (<b>27</b>), which was generated in excellent yield
by oxidation of the aldoxime precursor <b>26</b>. SuzukiāMiyaura
coupling of <b>27</b> with a variety of benzaldehyde 2-boronates
was accompanied by an aldol condensation/elimination reaction to give
the desired phenanthrene intermediate directly. Deprotection of the
benzyl alcohol followed by two sequential oxidation steps gave the
desired phenanthrene nitrocarboxylic acids. This approach was used
to synthesize AAs IāIV and several other related compounds,
including AA I and AA II bearing an aminopropyloxy group at position-6,
which were required for further conversion to fluorescent biological
probes. Further successful application of the SuzukiāMiyaura
coupling reaction to the synthesis of the <i>N-</i>hydroxyaristolactams
of AA I and AA II then allowed the synthesis of the putative, but
until now elusive, <i>N-</i>acetoxy- and <i>N-</i>sulfonyloxy-aristolactam metabolites