Mass spectrometric analysis of 2-deoxyribonucleoside and 2'-deoxyribonucleotide adducts with aldehydes derived from lipid peroxidation

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.     

Medial Prefrontal Lesions and Pavlovian Eyeblink and Heart Rate Conditioning: Effects of Partial Reinforcement on Delay and Trace Conditioning in Rabbits (Oryctolagus cuniculus).

Effects of continuous (100%) versus partial (25%) reinforcement were studied on Pavlovian delay and trace eyeblink conditioning in rabbits (Oryctolagus cuniculus) with either lesions to the medial prefrontal cortex (mPFC) or sham lesions. Concomitant heart rate changes evoked by the conditioned stimulus were also assessed. Partial reinforcement retarded eyeblink conditioning in both the trace and delay paradigm, but this impairment was greater during trace conditioning and in rabbits with mPFC lesions. Accompanying conditioned stimulus-evoked heart rate slowing was attenuated under all conditions by the mPFC lesions, although this result was not always statistically significant. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.     

Quantitative analysis of etheno-2'-deoxycytidine DNA adducts using on-line immunoaffinity chromatography coupled with LC/ES-MS/MS detection

Etheno DNA adducts, including 3,N4-etheno-2'-deoxycytidine (etheno-dC), are promutagenic lesions present in normal animal and human tissues. These DNA adducts are believed to be important in the etiology of cancer. Existing methods for quantifying etheno-dC use 32p. postlabeling. Although highly sensitive, postlabeling requires the use of an energetic radioisotope and considerable time and effort. The new methodology reported here permits automated quantification of trace levels of etheno-dC in crude DNA hydrolysates on the order of 5 adducts in 10(8) normal nucleotides from 100-microg samples of DNA. This was accomplished by using on-line immunoaffinity chromatography, a reverse-phase LC separation on graphitized carbon, tandem mass spectrometric detection, and an isotopically labeled internal standard. The automated procedures permitted analysis of 4 DNA hydrolysates/hr. The sensitivity using immunoaffinity cleanup was approximately 100-fold greater than that observed when using a silica-based trapping system. The validated method was applied to the analysis of etheno-dC in commercial calf thymus DNA, untreated mouse liver, and untreated rat liver DNA. The demonstrated level of performance suggests future applicability of this method in studies of cancer in humans and experimental animals.     

Interactions between the prefrontal cortex and amygdala during delay discounting and reversal.

Interactions between the prefrontal cortex and amygdala are thought to be critical for reward anticipation. Alterations in reward anticipation that lead to an inability to wait for rewards or a diminished capacity to change behavior when doing so would be optimal are often termed impulsivity and compulsivity, respectively. Distinct regions of the prefrontal cortex may support decreased impulsivity through self-control and decreased compulsivity through flexibility. However, both self-control and flexibility appear to involve the amygdala. Using a delay discounting paradigm, the current investigation found that inactivation and disconnection of the medial prefrontal cortex and basolateral amygdala led rats to become more impulsive by affecting preference for smaller immediate over larger delayed rewards. Conversely, inactivation and disconnection of the orbitofrontal cortex and amygdala led rats to become more compulsive as demonstrated by an inability to flexibly reverse stimulus–reward relationships in an odor reversal task. The current findings support a double dissociation between orbitofrontal cortex–amygdala interactions for odor reversal and medial prefrontal cortex–amygdala interactions for delay discounting. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mechanism for inhibition of thyroid peroxidase by leucomalachite green

The triphenylmethane dye, malachite green (MG), is used to treat and prevent fungal and parasitic infections in the aquaculture industry. It has been reported that the reduced metabolite of MG, leucomalachite green (LMG), accumulates in the tissues of fish treated with MG. MG is structurally related to other triphenylmethane dyes (e.g., gentian violet and pararosaniline) that are carcinogenic in the liver, thyroid, and other organs of experimental animals. The ability of LMG to inhibit thyroid peroxidase (TPO), the enzyme that catalyzes the iodination and coupling reactions required for thyroid hormone synthesis, was determined in this study. LMG inhibited TPO-catalyzed tyrosine iodination (half-maximal inhibition at ca. 10 microM). LMG also inhibited the TPO-catalyzed formation of thyroxine in low-iodine human goiter thyroglobulin (half-maximal inhibition at ca. 10 microM) using a model system that measures simultaneous iodination and coupling. Direct inhibition of the coupling reaction by LMG was shown using a coupling-only system containing chemically preiodinated thyroglobulin as the substrate. Incubation of LMG with TPO, iodide, and tyrosine in the presence of a H2O2-generating system yielded oxidation products that were identified by using on-line LC/APCI-MS as desmethyl LMG, 2desmethyl LMG, 3desmethyl LMG, MG, and MG N-oxide. Similar products from LMG were observed in incubations with TPO and H2O2 alone. These findings suggest that the anti-thyroid effects (increased serum thyroid-stimulating hormone and decreased serum thyroxine) observed in rats treated with LMG result from blockade of hormone synthesis through alternate substrate inhibition and that chronic exposure could cause thyroid follicular cell tumors through a hormonal mechanism. The observed TPO-catalyzed oxidative demethylation of LMG to a primary arylamine also suggests a genotoxic mechanism for tumor formation is possible.     

Positron emission tomographic imaging of the cardiovascular system: an emerging clinical tool

Cyclic GMP-dependent protein kinase (cGMP kinase) is the major receptor protein for cGMP in vascular smooth muscle. Vascular smooth muscle cells (VSMC) isolated from the rat aorta express type I cGMP kinase at high levels, but expression decreases markedly upon passage of the cells. In primary or early passage, the expression of cGMP kinase is lowest when cells are plated at low density as assessed by immunological and Northern analyses. Expression increases at confluence and is maintained in postconfluent cultures. With repeated passaging, however, the levels of cGMP kinase decrease even in confluent and postconfluent cultures so that after several passages enzyme levels are undetectable. The decrease in expression in passaged cells is not due to exposure to serum-derived growth factors, but rather on the repeated exposure of cells to conditions in which cell density is reduced (i.e., subculturing). These results indicate that aortic VSMC grown at low density or those repetitively passaged have reduced expression of cGMP kinase, and thus may not represent appropriate cultures with which to investigate the role of nitric oxide and cGMP in VSMC function.     

Medial prefrontal cortex and Pavlovian conditioning: Trace versus delay conditioning.

Pavlovian eyeblink (EB) conditioning was studied in both trace and delay paradigms in rabbits (Oryctolagus cuniculus) with either medial prefrontal cortex (mPFC) lesions or sham lesions. mPFC lesions of prelimbic cortex (Brodmann's Area 32) retarded EB conditioning in the trace but not the delay paradigm. However, this effect was significant only when the conditioned stimulus (CS) was 500 rather than 100 ms in duration. Lesions of the anterior cingulate cortex (Area 24) did not affect EB conditioning in a trace paradigm. Accompanying CS-evoked heart rate slowing was attenuated under all conditions by the mPFC lesions, although this result was not always statistically significant. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Posttraining lesions of the medial prefrontal cortex impair performance of Pavlovian eyeblink conditioning but have no effect on concomitant heart rate changes in rabbits (Oryctolagus cuniculus).

The medial prefrontal cortex (mPFC) plays a critical role in conditioned autonomic adjustments but is not involved in classically conditioned somatomotor responses unless the training conditions include reversal or trace conditioning. The studies showing these effects have all used pretraining lesions. The present study assessed the effects of posttraining lesions on eyeblink (EB) and heart rate (HR) conditioned responses (CRs) in both delay and trace conditioning paradigms in the rabbit (Oryctolagus cuniculus). Posttraining lesions lowered the percentage of EB CRs during retesting compared with pretesting levels for both delay and trace conditioning. Control lesions and pretraining lesions produced no significant effects during retesting. Posttraining lesions had no effect on the HR CR. These findings suggest that a critical mechanism in the mPFC is involved in retrieval of information during EB conditioning but that the mPFC integration of autonomic and somatomotor processes is not critical to this retrieval process. (PsycINFO Database Record (c) 2010 APA, all rights reserved)