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 A : T to T : A 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

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 ∼50% 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-dA→T and AL-II-dG→T transversions

Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence

Comparison of phenol red and polyethyleneglycol as nonabsorbable markers for the study of intestinal absorption in humans

When phenol red and polyethyleneglycol were used simultaneously as nonabsorbable markers in perfusion studies of the absorptive capacity of high jejunum in humans, apparent absorption was the same when calculated from either marker. This similar indication of dilution and of absorption by the two markers was found in normal subjects and in patients with nontropical sprue, whether aqueous or saline solutions of dextrose were infused. The similarity strengthens the evidence that either phenol red or polyethyleneglycol is a satisfactory “nonabsorbable” marker compound to indicate dilution in perfusion studies of dextrose and electrolyte absorption in limited segments of human intestine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44368/1/10620_2005_Article_BF02233070.pd