56 research outputs found
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
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
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
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
Structural and biochemical impact of C8-aryl-guanine adducts within the Narl recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity
Sherpa Romeo green journal, open accessChemical 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 carbonlinked
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
structures.Ye
An update on direct genotoxicity as a molecular mechanism of ochratoxin a carcinogenicity
Ochratoxin A (OTA) is a naturally occurring chlorophenolic fungal toxin that contaminates a wide range of food products and poses a cancer threat to humans. The mechanism of action (MOA) for OTA renal carcinogenicity is a controversial issue. In 2005, direct genotoxicity (covalent DNA adduct formation) was proposed as a MOA for OTA mediated carcinogenicity [Manderville, R. A. (2005) Chem. Res. Toxicol. 18, 1091-1097]. At that time, inconsistent results had been published on OTA genotoxicity/mutagenicity, and conclusive evidence for OTA-mediated DNA adduction had been lacking. In this update, published data from the past 6-7 years are presented that provide new hypotheses for the MOA of OTA-mediated carcinogenicity. While direct genotoxicity remains a controversial issue for OTA, new findings from the Umemura and Nohmi laboratories provide definitive results for the mutagenicity of OTA in the target tissue (outer medulla) of male rat kidney that rules out oxidative DNA damage. These findings, coupled with our own efforts that provide new structural evidence for DNA adduction by OTA, has strengthened the argument for involvement of direct genotoxicity in OTA-mediated renal carcinogenesis. This MOA should be taken into consideration for OTA human risk assessment
DNA Aptamer–Target Binding Motif Revealed Using a Fluorescent Guanine Probe: Implications for Food Toxin Detection
DNA aptamers are single-stranded
oligonucleotides that are generated
by an in vitro selection method to bind targets with high affinity
and specificity. Understanding molecular recognition by DNA aptamers
is of fundamental importance in the development of biosensor applications.
The small molecule ochratoxin A (OTA) is a fungal-derived food toxin,
and OTA DNA aptamers have been established for the development of
rapid detection platforms required for food safety. One such OTA aptamer
(OTAA) is a guanine-rich DNA oligonucleotide that folds into an antiparallel
G-quadruplex (GQ) upon OTA binding, although structural details of
the GQ fold and its interaction with OTA are currently unknown. In
the present study, the fluorescent nucleobase analogue, 8-thienyl-2′-deoxyguanosine
(ThdG), was inserted into various G sites of OTAA to determine the
probe impact on GQ folding and OTA binding affinity. Our results suggest
that OTAA contains three lateral (l) loops connecting two stacked
G-tetrads with an anticlockwise loop progression to afford a −(lll)
GQ topology. The phenolic ring system of OTA undergoes π-stacking
interactions with the G-tetrads of OTAA. Our results also demonstrate
aptamer sites that can be modified with ThdG to afford a fluorescent
light-up signal upon OTA binding
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