Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensity

Sherpa Romeo green journal, open accessThe nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2 -deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the associated adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to determine the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helices were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic separation between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixture of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity andmutagenic outcomes, and support recent experiments suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochemical experiments on this potentially genotoxic lesion.Ye

Ochratoxin A: In Utero Exposure in Mice Induces Adducts in Testicular DNA

Ochratoxin A (OTA) is a nephrotoxin and carcinogen that is associated with Balkan endemic nephropathy and urinary tract tumors. OTA crosses the placenta and causes adducts in the liver and kidney DNA of newborns. Because the testis and kidney develop from the same embryonic tissue, we reasoned that OTA also may cause adducts transplacentally in the testis. We tested the hypothesis that acute exposure to OTA, via food and via exposure in utero, causes adducts in testicular DNA and that these lesions are identical to those that can be produced in the kidney and testis by the consumption of OTA. Adult mice received a single dose of OTA (from 0–1,056 µg/kg) by gavage. Pregnant mice received a single i.p. injection of OTA (2.5 mg/kg) at gestation day 17. DNA adducts were determined by 32P-postlabeling. Gavage-fed animals sacrificed after 48 hours accumulated OTA in kidney and testis and showed DNA adducts in kidney and testis. Some OTA metabolites isolated from the tissues were similar in both organs (kidney and testis). The litters of mice exposed prenatally to OTA showed no signs of overt toxicity. However, newborn and 1-month old males had DNA adducts in kidney and testis that were chromatographically similar to DNA adducts observed in the kidney and testis of gavage-fed adults. One adduct was identified previously as C8-dG-OTA adduct by LC MS/MS. No adducts were observed in males from dams not exposed to OTA. Our findings that in utero exposure to OTA causes adducts in the testicular DNA of male offspring support a possible role for OTA in testicular cancer

Response to Comments of Peter G. Mantle

The apparently high yield of testis tumors (25%) in rats exposed long-term to Ochratoxin A (OTA) is uninterpretable without data on tumor yield in unexposed rats. Conversely, our demonstration that prenatal exposure to OTA induces DNA adducts in the testes of newborn mice and the absence of these adducts in the testes of mice not exposed prenatally to OTA, is evidence for the presumptive carcinogenicity of OTA in the testis. Together with recent data showing that prenatal exposure to OTA depresses expression of DMRT1, a tumor suppressor gene in the testis, our findings suggest that OTA may be a cause of testicular cancer

News Article on the Emancipations of 7 Enslaved Persons Named Minerva, Fanny, Auguste, Louis, Louisa, Victoire, and Zabette

News article of the report and resolution for the emanciation of 7 enslaved persons named Minerva, Fanny, Auguste, Louis, Louisa, Victoire, and Zabette, belonging to various enslavershttps://scholarsjunction.msstate.edu/lantern-um/1113/thumbnail.jp

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G3-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo− (Kf−) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structure

Mutagenicity of Ochratoxin A and Its Hydroquinone Metabolite in the SupF Gene of the Mutation Reporter Plasmid Ps189

Ochratoxin A (OTA) is a mycotoxin that enhances renal tumor formation in the outer medulla of male rat kidney. Direct DNA damage and subsequent mutagenicity may contribute to these processes. In this study we have determined whether OTA in the absence or presence of activated rat liver microsomes (RLM) or redox-active transition metals (Fe(III) or Cu(II)) causes promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. In addition, we have assessed the mutagenicity of the hydroquinone metabolite (OTHQ) of OTA in the absence or presence of cysteine without added cofactors. Our results show that oxidation of OTA, either by RLM or by transition metal ions, activates OTA to a directly genotoxic mutagen(s). The Fe(III)/OTA system was the most potent mutagen in our experimental system, causing a 32-fold increase in mutant fraction (MF) above the spontaneous control MF. The Cu(II)/OTA system caused a 9-fold increase in MF, while a 6–10-fold increase in MF was observed for OTA in the presence of RLM. The OTHQ metabolite is also mutagenic, especially in the presence of cysteine, in which a 6-fold increase in MF was observed. Our data provide further insight into OTA bioactivation that may account for its in vivo mutagenicity in male rat kidney

Development and Characterization of a Monoclonal Antibody against Ochratoxin B and Its Application in ELISA

A monoclonal antibody specific to ochratoxin B (OTB) was employed for the development of an indirect competitive OTB-ELISA. The optimized OTB-ELISA resulted in a limit of detection (LOD) for OTB of 3 µg/L (8 nM), a limit of quantification (LOQ) of 3.7 µg/L (10 nM), and a 50% inhibitory concentration (IC50) of 150 nM. Due to very low cross-reactivity to OTA (2.7%) and structurally related molecules (0%), this OTB-ELISA was found to be suitable to detect OTB with excellent precision in different matrices, i.e., beer, coffee and wine. Therefore, this OTB-ELISA will allow screening of OTB in food and feed products

Glutationski konjugati okratoksina A kao biomarkeri izloženosti

In the present study the photoreactivity of the fungal carcinogen ochratoxin A (OTA) has been utilised to generate authentic samples of reduced glutathione (GSH) and N-acetylcysteine (NAC) conjugates of the parent toxin. These conjugates, along with the nontoxic OTα, which is generated through hydrolysis of the amide bond of OTA by carboxypeptidase A, were utilised as biomarkers to study the metabolism of OTA in the liver and kidney of male and female Dark Agouti rats. Male rats are more susceptible than female rats to OTA carcinogenesis with the kidney being the target organ. Our studies show that the distribution of OTA in male and female rat kidney is not significantly different. However, the extent of OTA metabolism was greater in male than female rats. Much higher levels of OTα were detected in the liver compared to the kidney, and formation of OTα is a detoxification pathway for OTA. These findings suggest that differences in metabolism between male and female rats could provide an explanation for the higher sensitivity of male rats to OTA toxicity.U ovom je ispitivanju korištena fotoreaktivnost kancerogenog mikotoksina okratoksina A (OTA) kako bi se stvorili izvorni uzorci reduciranih glutationskih (GSH) i N-acetilcisteinskih (NAC) konjugata osnovnog toksina. Ovi konjugati, uz netoksični OTα, koji se stvara hidrolizom amidne veze OTA putem karboksipeptidaze A, upotrijebljeni su kao biomarkeri za ispitivanje metabolizma OTA u jetri i bubregu ženki i mužjaka štakora soja Dark Agouti. Mužjaci su se pokazali podložnijima stvaranju bubrežnih tumora uzrokovanih OTA toksinom od ženki. Utvrdili smo da se raspodjela OTA u bubrezima ženki i mužjaka značajno ne razlikuje. Međutim mužjaci su imali intenzivniji metabolizam OTA nego ženke. U jetri su utvrđene mnogo više razine OTα u usporedbi s bubregom, a rezultati upućuju na to da je stvaranje OTα detoksifi kacijski put za OTA. Zaključujemo da bi se veća osjetljivost mužjaka štakora na toksičnost OTA mogla pripisati spolno uvjetovanim razlikama u njegovu metabolizmu