Richard A. Manderville

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.

Copper-nuclease efficiency correlates with cytotoxicity for the 4-methoxypyrrolic natural products

The DNA-targeting activities of the 4-methoxypyrrolic natural products, that include prodigiosin (1), tambjamine E (2), and the blue pigment (3), have been compared using fluorescence spectroscopy to study DNA binding and agarose gel electrophoresis to assess their ability to facilitate oxidative copper-promoted DNA cleavage. Fluorescence emission titration of 3 with calf-thymus DNA (CT-DNA) shows that the natural product occupies a site size (n) of ca. two base pairs and possesses an affinity constant (K) of approximately 6x10(5) x M(-1). Similar to prodigiosin (1), the blue pigment 3 was found to facilitate oxidative double-strand DNA (dsDNA) cleavage without the aid of an external reducing agent. Quantitation of ds- (n2) and ss- (n1) breaks provided n1:n2 ratios of approximately 8-12, which were significantly greater than the number expected from the accumulation of ss-breaks (approximately 120). This was contrasted by the nicking activity of tambjamine E (2), which only generates ss-breaks in the presence of copper. The superior copper-nuclease activity of 1 and 3 also correlated with their superior anticancer properties against leukemia (HL-60) cells. These results are discussed with respect to the mode of cytotoxicity by the 4-methoxypyrrolic natural products.

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.

Structure–activity relationships for the fluorescence of ochratoxin A: Insight for detection of ochratoxin A metabolites

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium that is widely found as a contaminant of food products. The toxin is a renal carcinogen in male rats, the cause of mycotoxicoses in pigs and has been associated with chronic human kidney diseases. Bioactivation has been implicated in OTA-mediated toxicity, although inconsistent results have been reported, due, in part, to the difficulty in detecting OTA metabolites in vivo. Liquid chromatography (LC) coupled with fluorescence detection (FLD) is the most widely used analytical detection method for OTA. Under acidic conditions the toxin generates blue fluorescence (465 nm) that is due to an excited state intramolecular proton transfer (ESIPT) process that generates an emissive keto tautomer. Disruption of this ESIPT process quenches fluorescence intensity and causes a blue shift in emission maxima. The aim of the present study was to determine the impact of the C5-chlorine atom, the lactone moiety and the amide bond on OTA fluorescence and derive optical parameters for OTA metabolites that have been detected in vitro. Our results highlight the limitations of LC/FLD for OTA metabolites that do not undergo ESIPT. For emissive derivatives, our absorption and emission data improves the sensitivity of LC/FLD (3-4-fold increase in the limit of detection (LOD)) for OTA analogues bearing a C5-OH group, such as the hydroquinone (OTHQ) metabolite and the glutathione conjugate of OTA (OTA-GSH). This increased sensitivity may facilitate the detection of OTA metabolites bearing a C5-OH group in biological fluids and enhance our understanding of OTA-mediated toxicity.

Glutathione Conjugates of Ochratoxin a as Biomarkers of Exposure / Glutationski Konjugati Okratoksina A Kao Biomarkeri Izloženosti

Abstract 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 detoksifikacijski put za OTA. Zaključujemo da bi se veća osjetljivost mužjaka štakora na toksičnost OTA mogla pripisati spolno uvjetovanim razlikama u njegovu metabolizmu.

Electronic tuning of fluorescent 8-aryl-guanine probes for monitoring DNA duplex–quadruplex exchange

In DNA-based diagnostics, duplex–quadruplex exchange is a common strategy for target detection using fluorescent probes that turn-on during the exchange process. Typical “label” detection platforms use emissive tags that are attached via linkers to the 5′- or 3′-ends of the oligonucleotide. Alternatively, “label-free” strategies employ fluorescent molecules that bind specifically to G-quadruplex structures with enhanced emission. Here, we report the utility of two internal fluorescent 8-aryl-2′-deoxyguanine probes (8-furyl-dG (FurdG) and 8-(4′′-cyanophenyl)-dG (CNPhdG)) for detecting G-quadruplex folding by the 15-mer (5′-GGTTG5G6TG8TGGTTGG) thrombin-binding aptamer (TBA). The 8-aryl-dG probes adopt a syn-conformation and were inserted into G5 (syn), G6 (anti) and G8 (TGT loop) of TBA to study their site-specific impact on duplex and G-quadruplex folding. Our studies show the ability of 8-aryl-dG probes to preferentially stabilize the G-quadruplex structure of TBA at G5 and their acceptance within the TG8T loop despite their syn-preference. Our studies also demonstrate how the choice of 8-aryl substituent can be used to tune probe electronics for turn-on fluorescence in the duplex or G-quadruplex structure. Overall, our studies establish 8-aryl-dG probes as useful tools for DNA-based diagnostics.

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

The 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 and mutagenic 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.

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 structures.

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.

Ochratoxin A acts as a photoactivatable DNA cleaving agent

The ability of ochratoxin A to photoinduce DNA cleavage is described; in the presence of DNA the photoreaction yields the non-chlorinated derivative, ochratoxin B, while a hydroquinone derivative is produced under anaerobic conditions.