Glutathione conjugates of ochratoxin A as biomarkers of exposure.

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.

Structure-activity relationships imply different mechanisms of action for ochratoxin A-mediated cytotoxicity and genotoxicity.

Ochratoxin A (OTA) is a fungal toxin that is classified as a possible human carcinogen based on sufficient evidence for carcinogenicity in animal studies. The toxin is known to promote oxidative DNA damage through production of reactive oxygen species (ROS). The toxin also generates covalent DNA adducts, and it has been difficult to separate the biological effects caused by DNA adduction from that of ROS generation. In the current study, we have derived structure-activity relationships (SAR) for the role of the C5 substituent of OTA (C5-X = Cl) by first comparing the ability of OTA, OTBr (C5-X = Br), OTB (C5-X = H), and OTHQ (C5-X = OH) to photochemically react with GSH and 2'-deoxyguanosine (dG). OTA, OTBr, and OTHQ react covalently with GSH and dG following photoirradiation, while the nonchlorinated OTB does not react photochemically with GSH and dG. These findings correlate with their ability to generate covalent DNA adducts (direct genotoxicity) in human bronchial epithelial cells (WI26) and human kidney (HK2) cells, as evidenced by the (32)P-postlabeling technique. OTB lacks direct genotoxicity, while OTA, OTBr, and OTHQ act as direct genotoxins. In contrast, their cytotoxicity in opossum kidney epithelial cells (OK) and WI26 cells did not show a correlation with photoreactivity. In OK and WI26 cells, OTA, OTBr, and OTB are cytotoxic, while the hydroquinone OTHQ failed to exhibit cytotoxicity. Overall, our data show that the C5-Cl atom of OTA is critical for direct genotoxicity but plays a lesser role in OTA-mediated cytotoxicity. These SARs suggest different mechanisms of action (MOA) for OTA genotoxicity and cytotoxicity and are consistent with recent findings showing OTA mutagenicity to stem from direct genotoxicity, while cytotoxicity is derived from oxidative DNA damage.

Concerning the hydrolytic stability of 8-aryl-2'-deoxyguanosine nucleoside adducts: implications for abasic site formation at physiological pH.

Direct addition of aryl radical species to the C(8)-site of 2'-deoxyguanosine (dG) affords C(8)-aryl-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons (PAHs), and certain phenolic toxins. A common property of C(8)-arylpurine adduction is the accompaniment of abasic site formation. To determine how the C(8)-aryl moiety contributes to sugar loss, UV-vis spectroscopy has been employed to determine N(7) pK(a1) values and hydrolysis kinetics, while density functional theory (DFT) calculations have been utilized to probe the structural features and stability of the C(8)-aryl-dG adducts bearing different para and ortho substituents. In all cases, the C(8)-aryl-dG adducts adopt a syn conformation containing a strong O(5)'-H...N(3) hydrogen bond with the aryl ring twisted with respect to the nucleobase. The adducts undergo N(7)-protonation with ionization constants and calculated N(7) proton affinity (PA) values similar to those measured for dG. The hydrolysis kinetics shows that C(8)-aryl-dG nucleoside adducts are more prone than dG to acid-catalyzed hydrolysis, with those bearing para substituents having k(1) values that are ca. 90- to 200-fold larger than k(1) for dG, while the effects for the ortho adducts are only ca. 9- to 60-fold larger. Changes in the rate of hydrolysis are further explained by calculations showing that glycosidic bond cleavage in the syn orientation of both neutral and N(7)-protonated dG has a lower barrier than the anti orientation, and the bulky (phenyl) group further decreases the barrier. Despite adduct reactivity in acidic media, all adducts are relatively stable at physiological pH with t(1/2) approximately 25 days, suggesting that they are unlikely intermediates leading to abasic site formation at physiological pH. This information has allowed development of a new rationale for the tendency of abasic site formation to accompany C(8)-arylpurine adduction within duplex DNA at neutral pH.

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.