Roger A. Coulombe

DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: Structure-activity relationships

Pyrrolizidine alkaloids (PAs) are common constituents of many species of flowering plants which possess carcinogenic as well as anticarcinogenic activity in vivo. Pyrrolizidine alkaloids are genotoxic in various short-term assays. The mechanisms by which these compounds exert these effects is still unclear. In this study, we characterized the ability of eight bifunctional PAs, with differing stereochemistry and functional groups, to cross-link cellular DNA in cultured bovine kidney epithelial cells. PAs representative of three major structural classes, the macrocycles (seneciphylline, riddelline, retrorsine, senecionine, monocrotaline), the open diesters (heliosupine, latifoline), and pyrrolizidine base (retronecine) were cultured for 2 hr with cells and an external metabolizing system. Every PA induced DNA cross-links which consisted primarily of proteinase-sensitive cross-links (DPC), but also to a smaller extent, DNA interstrand cross-links (ISC). None of the PAs induced detectable amounts of DNA single-strand breaks. The PAs which produced DPC and/or ISC (ranked from most potent to least) were: seneciphylline (DPC greater than ISC); riddelline (DPC greater than ISC); retrorsine (DPC greater than ISC); senecionine (DPC greater than ISC); heliosupine (DPC greater than ISC); monocrotaline (ISC = DPC); latifoline (DPC greater than ISC); and retronecine (ISC greater than DPC). Although the PAs induced DNA cross-linking to varying degrees, cell viabilities for all treatment groups were greater than 90% as determined by trypan blue dye exclusion. Since the cross-linking ability of these PAs paralleled their ability to inhibit colony formation, cross-link formation may be involved in the biological activity of these compounds. Two structural determinants of biological activity appear to be the presence of both a macrocyclic necic acid ester and an alpha,beta-unsaturated ester function since the cross-linking ability of seneciphylline, riddelline, retrorsine, and senecionine far exceeded that of monocrotaline, heliosupine, latifoline, and retronecine. In addition, the stereochemical orientation of the ester linkage was found to have no effect on biological activity.

Effects of PM2.5 collected from Cache Valley Utah on genes associated with the inflammatory response in human lung cells.

In January 2004, the normally picturesque Cache Valley in northern Utah made national headlines with the highest PM2.5 levels in the nation. Epidemiological studies linked exposure to particulate air pollution in other locations with stroke and Alzheimers disease and to early mortality from all causes, cancer, and cardiopulmonary diseases. To determine potential effects of these particles on human health, human bronchial epithelial cells (BEAS-2B) were cultured with PM2.5 collected from various locations in the Cache Valley. These particles were slightly cytotoxic, but more potent than NH4NO3, the major chemical component of Cache Valley PM2.5. Gene expression analysis of PM2.5-exposed cells was performed using microarray and quantitative reverse-transcription polymerase chain reaction (RT-PCR.) Among other genes, PM2.5 exposure induced genes and proteins involved in the inflammatory response. Most notably, PM2.5-exposed cells showed significant gene level upregulation of activating receptors to interleukins 1 and 6 (IL-1R1 and IL-6R), as well as concomitant increases in protein. Increases in IL-1 receptor associated kinase-1 (IRAK) protein were observed. PM2.5 exposure resulted in release of IL-6, as well phosphorylated STAT3 protein, providing evidence that PM activates the IL-6/gp130/STAT3 signaling pathway in BEAS-2B cells. IL-20 and major histocompatibility complex peptide class-1 (MICA) were upregulated and cleavage of caspase-12 was detected. In total, our results indicate that Cache Valley PM2.5 produces the upregulation of important cytokine receptors and is able to activate both IL-1R- and IL-6R-mediated signaling pathways in human lung cells. These observations are generally consistent with the adverse effects associated with inhalation of fine particulate matter like PM2.5.

Metabolism of aflatoxin B1 in Turkey liver microsomes: The relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B(1)-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB(1) to the exo-AFBO and to detoxification products aflatoxin M(1) (AFM(1)) and aflatoxin Q(1) (AFQ(1)), respectively, complicates the kinetic analysis of AFB(1) in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB(1) in TLMs. In control TLMs, AFB(1) was converted to exo-AFBO in addition to AFM(1) and AFQ(1) confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~0.1μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (>50μM) AFB(1) concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM(1), conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ(1) following the kinetic Hill equation. At 0.1μM AFB(1), close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM(1)) compared to 3A37 (0.15: 1, exo-AFBO: AFQ(1)), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB(1) bioactivation and metabolism at environmentally-relevant AFB(1) concentrations in turkey liver.

Importance of Hydroxyl Radical in the Vanadium-Stimulated Oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.

Effects of dietary vanadium exposure on levels of regional brain neurotransmitters and their metabolites

Adult male CD-1 mice were treated with various levels of vanadate in drinking water for 30 days. The levels of catecholamine and indoleamine neurotransmitters and their major metabolites were measured in six different brain regions. Vanadium caused a dose-related decrease in norepinephrine (NE) levels in hypothalamus, the region rich in this biogenic amine. Levels of the NE metabolite, vanillylmandelic acid (VMA), correspondingly decreased in the same region. Although hypothalamic dopamine (DA) also showed a significant decline, vanadium had little effect on DA metabolites. Levels of 5-hydroxytryptamine (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were not influenced. Levels of DA were not affected in the corpus striatum, where the highest levels of this amine are observed. Effects of vanadium on various biogenic amines and their metabolites were only marginal in other brain regions. Results suggest that vanadium has a selective effect on adrenergic pathways, and effects on other hypothalamic amines appear to be secondary. These observations support the pro-oxidant potential of vanadate ion on catecholamines suggested earlier.

Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster

Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B(1) (AFB(1)) and GST dysfunction is a known risk factor for susceptibility towards AFB(1). Turkeys are one of the most susceptible animals known to AFB(1), which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB(1)-8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the alpha-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four alpha-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic alpha-class GSTs in the turkey. Four signature motifs and conserved residues found in alpha-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the alpha-class GST gene cluster was isolated and sequenced. The turkey alpha-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human alpha-class GSTs and flanking genes. This study identifies the alpha-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB(1) susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway.

Immunological and neurobiochemical alterations induced by repeated oral exposure of phenol in mice

Phenol, a major metabolite of benzene, is a potentially immunotoxic and neurotoxic substance of environmental significance. Male CD-1 mice were continuously exposed to 0, 4.7, 19.5, and 95.2 mg phenol/l in drinking water for 4 weeks. Various immune functions were evaluated and levels of selected neurotransmitters and metabolites measured in discrete brain regions. The doses of phenol did not produce any overt clinical signs of toxicity; peripheral red blood cell counts and hematocrits decreased. A dose of 95.2 mg/l suppressed the stimulation of cultured splenic lymphocytes by lipopolysaccharide, pokeweed mitogen, and phytohemagglutinin and the response in mixed lymphocyte cultures. The two high doses suppressed antibody production response to the T cell-dependent antigen (sheep erythrocytes), as determined by plaque-forming cells, and serum antibody levels. Mice treated with phenol had lower levels of neurotransmitters in several brain regions. In the hypothalamus, a major norepinephrine-containing compartment, the concentrations of norepinephrine significantly decreased by 29 and 40% in groups dosed with 19.5 and 95.2 mg/l, while dopamine concentrations decreased in the corpus striatum by 21, 26, and 35% at 4.7, 19.5 and 95.2 mg/l, respectively. Phenol also decreased 5-hydroxytryptamine in the hypothalamus, medulla oblongata, midbrain and corpus striatum. Levels of monoamine metabolites decreased in the hypothalamus (5-hydroxyindoleacetic acid), midbrain (vanillylmandelic acid), corpus striatum (vanillylmandelic acid and dihydroxyphenylacetic acid), cortex (vanillylmandelic acid), and cerebellum (dihydroxyphenylacetic acid).

Evaluation of toluene exposure via drinking water on levels of regional brain biogenic monoamines and their metabolites in CD-1 mice

Toluene, a potentially neurotoxic substance, is found in trace amounts in groundwater. Adult male CD-1 mice were continuously fed drinking water ad libitum containing 0, 17, 80, and 405 mg/liter toluene. After a 28-day treatment, animals were tested for endogenous levels of the biogenic monoamines norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and their respective metabolites. 3-methoxy-4-hydroxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), in six discrete brain regions. The maximum toluene-induced increases of biogenic amines and their metabolites generally occurred at a toluene concentration of 80 mg/liter. In the hypothalamus, a major NE-containing compartment, the concentrations of NE significantly increased by 51, 63, and 34% in groups dosed with 17, 80, and 405 mg/liter, respectively. Significant increases of NE were also observed in the medulla oblongata and midbrain. Concomitantly, concentrations of VMA increased in various brain regions. Concentrations of DA were significantly higher in the corpus striatum and hypothalamus. Alterations in levels of DA metabolites, DOPAC and HVA, were marginal. Toluene significantly increased concentrations of 5-HT in all dissected brain regions, except cerebellum, and increased the 5-HIAA levels in the hypothalamus, corpus striatum, and cerebral cortex.

Aflatoxicosis chemoprevention by probiotic Lactobacillius and lack of effect on the major histocompatibility complex

Turkeys are extremely sensitive to aflatoxin B1 (AFB1) which causes decreased growth, immunosuppression and liver necrosis. The purpose of this study was to determine whether probiotic Lactobacillus, shown to be protective in animal and clinical studies, would likewise confer protection in turkeys, which were treated for 11 days with either AFB1 (AFB; 1 ppm in diet), probiotic (PB; 1 × 10(11) CFU/ml; oral, daily), probiotic + AFB1 (PBAFB), or PBS control (CNTL). The AFB1 induced drop in body and liver weights were restored to normal in CNTL and PBAFB groups. Hepatotoxicity markers were not significantly reduced by probiotic treatment. Major histocompatibility complex (MHC) genes BG1 and BG4, which are differentially expressed in liver and spleens, were not significantly affected by treatments. These data indicate modest protection, but the relatively high dietary AFB1 treatment, and the extreme sensitivity of this species may reveal limits of probiotic-based protection strategies.

Vanadate Stimulation of Pyridine Nucleotide Oxidation in Mammalian Liver Microsomal Membranes

Vanadate has been shown in numerous experiments to markedly stimulate the oxidation of NAD(P)H in biological membranes. However, explanations of the nature of this stimulation often have been contradictory. Vanadate-catalyzed oxidations have been postulated to be a result of a specific membrane-associated NADH oxidase.1 Liochev and Fridovich2 have reported that this phenomenon reflects a vanadate stimulation of NAD(P)H oxidation by superoxide rather than by vanadate-stimulated NAD(P)H oxidases or dehydrogenases. The purpose of the present study was to characterize the nature of the vanadate stimulation of pyridine nucleotide oxidation in membranes from rat liver and to determine if vanadate is capable of stimulating NADPH oxidation in systems containing purified NADPH cytochrome P-450 reductase.