Analysis of cellular 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 ± 0.4 8-oxo-dGuo/107 dGuo (mean ± standard deviation) or 5.5 ± 1.0 8-oxo-dGuo/108 nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose−response for 8-oxo-dGuo formation (r2 = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

Eighteen primiparous Holstein cows were used in a 10-wk lactation study, preceded by a 2-wk covariate period, to determine the effect of concentration of deoxynivalenol in the diet on cow performance and transfer of deoxynivalenol and its metabolite, deepoxydeoxynivalenol, to milk. Diets were formulated to contain deoxynivalenol at 0, 6, and 12 mg/kg of concentrate DM, and daily intake of deoxynivalenol was .59, 42, and 104 mg, respectively. Increasing deoxynivalenol in the diet did not affect intake of concentrate or forage. Total milk output was not affected; however, milk fat responded quadratically; cows given deoxynivalenol at 6 mg/kg of concentrate DM had the lowest milk fat content and fat output. Overall energetic efficiency was not influenced because reduced energy output in milk was compensated by increased BW gains. No transfer of deoxynivalenol or deepoxydeoxynivalenol to milk was observed; concentrations were below detectable limits (1 microgram/ml) using HPLC-mass spectroscopy. We concluded that diets containing deoxynivalenol up to 6 mg/kg of dietary DM did not reduce feed intake of cows in this study and that deoxynivalenol or deepoxydeoxynivalenol was not transferred to milk. Further studies are required to confirm the apparent lack of effect of deoxynivalenol on milk production.

A quick and easy method for the analysis of anticoagulant rodenticides in blood or tissue using principles of dispersive solid-phase extraction (dSPE), commonly known as QuEChERS (short for quick, easy, cheap, effective, rugged, and safe), was developed. Briefly, a combination of magnesium sulfate, PSA, florisil, and basic alumina was used to cleanup blood samples. Further, to cleanup liver tissue samples, C(18) sorbent was included along with the previously mentioned. The samples were analyzed using high-performance liquid chromatography equipped with a reversed-phase C(18) column (150 x 4.6 mm, 5-microm particle size) and a UV and fluorescence detector. The mobile phase consisted of 0.03 M tetrabutylammonium hydroxide (TBA) adjusted to pH 7/methanol (1:1, v/v) as solvent A and methanol as solvent B in a gradient run. The method detection limit was as low as 10 ng/mL for brodifacoum and difenacoum in blood and 10 ng/g in liver; 50 ng/mL for bromadiolone, difethialone, and chlorphacinone in blood and similarly 50 ng/g in liver; and 100 ng/mL for coumafuryl, pindone, warfarin, and diphacinone in blood and 100 ng/g in liver samples. A number of clinical samples of both blood and liver were analyzed; the comparison of this modified QuEChERS and traditional solid-phase extraction data was found to be in close agreement. This method resulted in drastic reduction in processing time and solvent cost both in terms of consumption and disposal, thus making it an attractive alternative to the traditional solid-phase extraction.

Benzo[a]pyrene (BP) requires metabolic activation to electrophiles to exert its deleterious effects. We compared the respective roles of aldo-keto reductase 1A1 (AKR1A1, aldehyde reductase) and P4501B1 in the formation of BP-7,8-dione and BP-tetrols, respectively, in intact bronchoalveolar cells manipulated to express either enzyme. Metabolite formation was confirmed by HPLC/MS and quantitatively measured by HPLC/UV/beta-RAM. In TCDD-treated H358 cells (P4501B1 expression), the anti-BPDE hydrolysis product BP-tetrol-1 increased over 3-12 h to a constant level. In H358 AKR1A1 transfectants, formation of BP-7,8-dione was elevated for 3-12 h but significantly decreased after 24 h. Interestingly, BP-tetrols were also detected in AKR1A1 transfectants even though they do not constitutively express P4501A1/P4501B1 enzymes. Northern and Western blotting confirmed the induction of P4501B1 by BP-7,8-dione in parental cells and the induction of P4501B1 by BP-7,8-diol in AKR1A1-transfected cells. P4501B1 induction was blocked in AKR1A1 transfectants by the AKR1A1 inhibitor (sulfonylnitromethane), the o-quinone scavenger (N-acetyl-l-cysteine), or the cytosolic AhR antagonist (diflubenzuron). Attenuation of P4501B1 induction in these cells was verified by measuring a decrease in BP-tetrol formation. Our studies show that the formation of BP-7,8-dione by AKR1A1 in human bronchoalveolar cells leads to an induction of P4501B1 and that a functional consequence of this induction is elevated anti-BPDE production as detected by increased BP-tetrol formation. Therefore, the role of AKR1A1 in the activation of BP-7,8-diol is bifunctional; that is, it directly activates BP-7,8-diol to the reactive and redox-active PAH o-quinone (BP-7,8-dione) and it indirectly trans-activates the P4501B1 gene by generating the aryl hydrocarbon receptor (AhR) ligand BP-7,8-dione.

Presence of fumonisin B1 (FB1), a major metabolite of Fusarium moniliforme, in corn is of great concern to both human and animal health because of its wide range of toxicity. The pharmacokinetics of FB1 was studied in laying hens following oral and intravenous administration of 14C-labelled FB1. After iv dosing (2.0 mg = 23.68 kBq/kg bw) plasma radioactivity underwent a very rapid bi-exponential decline (t1/2 alpha = 2.5 +/- 0.3 min; t1/2 beta = 48.8 +/- 11.2 min) with negligible levels measured after 4-6 hr. Mean value for the apparent volume of distribution at steady state (Vdss) was 18.27 ml/kg, apparent volume of central compartment (Vd beta) was 82.20 ml/kg and plasma clearance was 1.18 ml/min/kg. At 24 hr post-dosing only trace residues were present in liver, kidney, and cecum. When dosed by the oral route (2.0 mg = 47.36 kBq/kg bw), systemic absorption of fumonisin appeared to be poor (F = 0.71 +/- 0.5%) with peak plasma concentrations of only 40-145 dpm/ml (equivalent to 28-103 ng FB1 and/or metabolites per ml) between 1.5 and 2.5 hr. At 24 hr post-dosing only trace amounts were present in crop, liver, kidney, small intestine, and cecum. In both orally and iv dosed birds almost all (97.7 +/- 3.73%) of the radioactivity was recovered in excreta by the end of the 24 hr experiment period and no residues were found in eggs laid during the 24 hr post-dosing period.(ABSTRACT TRUNCATED AT 250 WORDS)