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Lawrence J Marnett

Publications and source records attributed to Lawrence J Marnett.

At least 19 recordsLinked to original sources
PubMed2026

The Oxidative DNA Lesion 6-Oxo-M1dG is a Potent Replication Block, Inducing Deletions and Base Substitution Mutations In Vivo.

One of the most prevalent exocyclic DNA adducts is 3-(2-deoxy-β-D-erythro-pentofuranosyl) pyrimido[1,2-α]purin-10(3H)-one (M1dG), an oxidative DNA lesion that forms by the reaction of guanines in nucleic acids and pool nucleotides with oxidation-induced base propenals or with the lipid peroxidation product, malondialdehyde. Further oxidation converts M1dG to 6-oxo-M1dG, an even more deleterious lesion whose genotoxic and mutagenic properties have been characterized in vitro. The present work uses a site-specifically modified viral genome to evaluate the biochemical consequences of 6-oxo-M1dG in Escherichia coli cells and contrast them with the properties of the M1dG parent lesion. We found that 6-oxo-M1dG strongly inhibited replication, with a bypass efficiency of 1-2%, relative to an unmodified guanine. By contrast, under the same experimental conditions, the bypass efficiency of M1dG was 30-40%. Beyond its low bypass rate, 6-oxo-M1dG was 20 times more mutagenic than M1dG, with the majority of mutations being single base deletions. However, when the levels of bypass polymerases were increased by inducing the SOS response, the proportion of deletions decreased, at the expense of additional single base substitutions, primarily G → T and G → C mutations. Finally, two DNA repair pathways─the direct reversal dioxygenase AlkB and glycosylase MutY─were investigated for their putative activity on 6-oxo-M1dG. The results indicated that neither system was capable of repairing this highly mutagenic lesion.

Escherichia coli↗
PubMed2006

Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications.

Glycerophospholipids containing arachidonic acid (20:4) serve as the precursors for an array of biologically active lipid mediators, most of which are produced by macrophages. We have applied mass spectrometry-based lipid profiling technology to evaluate the glycerophospholipid structure and composition of two macrophage populations, resident peritoneal macrophages and RAW264.7 cells, with regard to their potential for 20:4-based lipid mediator biosynthesis. Fatty acid analysis indicated that RAW264.7 cells were deficient in 20:4 (10 +/- 1 mol %) compared to peritoneal macrophages (26 +/- 1 mol %). Mass spectrometry of total glycerophospholipids demonstrated a marked difference in the distribution of lipid species, including reduced levels of 20:4-containing lipids, in RAW264.7 cells compared to peritoneal macrophages. Enrichment of RAW264.7 cells with 20:4 increased the fatty acid to 20 +/- 1 mol %. However, the distribution of the incorporated 20:4 remained different from that of peritoneal macrophages. RAW264.7 cells pretreated with granulocyte-macrophage colony stimulating factor followed by lipopolysaccharide and interferon-gamma mobilized similar quantities of 20:4 and produced similar amounts of prostaglandins as peritoneal macrophages treated with LPS alone. LPS treatment resulted in detectable changes in specific 20:4-containing glycerophospholipids in peritoneal cells, but not in RAW264.7 cells. 20:4-enriched RAW264.7 cells lost 88% of the incorporated fatty acid during the LPS incubation without additional prostaglandin synthesis. These results illustrate that large differences in glycerophospholipid composition may exist, even in closely related cell populations, and demonstrate the importance of interpreting the potential for lipid-mediator biosynthesis in the context of overall glycerophospholipid composition.

Animals↗
PubMed2006

Targeted cyclooxygenase gene (ptgs) exchange reveals discriminant isoform functionality.

The prostaglandin G/H synthase enzymes, commonly termed COX-1 and COX-2, differ markedly in their responses to regulatory stimuli and their tissue expression patterns. COX-1 is the dominant source of "housekeeping" prostaglandins, whereas COX-2 synthesizes prostaglandins of relevance to pain, inflammation, and mitogenesis. Despite these distinctions, the two enzymes are remarkably conserved, and their subcellular distributions overlap considerably. To address the functional interchangeability of the two isozymes, mice in which COX-1 is expressed under COX-2 regulatory elements were created by a gene targeting "knock-in" strategy. In macrophages from these mice, COX-1 was shown to be lipopolysaccharide-inducible in a manner analogous to COX-2 in wild-type macrophages. However, COX-1 failed to substitute effectively for COX-2 in lipopolysaccharide-induced prostaglandin E2 synthesis at low concentrations of substrate and in the metabolism of the endocannabinoid 2-arachidonylglycerol. The marked depression of the major urinary metabolite of prostacyclin in COX-2 null mice was only partially rescued by COX-1 knock-in, whereas the main urinary metabolite of prostaglandin E2 was rescued totally. Replacement with COX-1 partially rescued the impact of COX-2 deletion on reproductive function. The renal pathology consequent to COX-2 deletion was delayed but not prevented, whereas the corresponding peritonitis was unaltered. Insertion of COX-1 under the regulatory sequences that drive COX-2 expression indicated that COX-1 can substitute for some COX-2 actions and rescue only some of the consequences of gene disruption. Manipulation of COX-2 also revealed a preference for coupling with distinct downstream prostaglandin synthases in vivo. These mice will provide a valuable reagent with which to elucidate the distinct roles of the COX enzymes in mammalian biology.

3' Untranslated Regions↗
PubMed2006

Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2.

COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.

Animals↗
PubMed2006

Nitric oxide deficiency promotes vascular side effects of cyclooxygenase inhibitors.

The cardiovascular safety of COX-2 selective and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) has recently been called into question. The factors that predispose to adverse events by NSAIDs are unknown. Because patients with arthritis have decreased nitric oxide (NO) bioavailability, the in vivo effects of NSAIDs on murine vascular tone and platelet activity in the presence or absence of NO were examined. Here, we show that acute hypertensive and prothrombotic activities of the COX-2-selective inhibitor celecoxib are revealed only after in vivo inhibition of NO generation. The nonselective NSAID indomethacin was hypertensive but antithrombotic when NO was absent. In vitro myography of aortic rings confirmed that vasoconstriction required inhibition of both NOS and COX-2 and was abolished by supplementation with exogenous NO. These data indicate that NO suppresses vascular side effects of NSAIDs, suggesting that risk will be greatest in patients with impaired vascular function associated with decreased NO bioavailability.

Animals↗
PubMed2006

Malondialdehyde, a major endogenous lipid peroxidation product, sensitizes human cells to UV- and BPDE-induced killing and mutagenesis through inhibition of nucleotide excision repair.

Aldehydes are ubiquitous contaminants in the human environment. Intracellular aldehydes are mainly derived from the metabolism of polyunsaturated fatty acids and from lipid peroxidation, which is significantly elevated under oxidative stress conditions. Oxidative stress has long been suspected to be involved in many disease processes, including carcinogenesis, neurodegeneration and aging, but its mechanisms are largely unknown. Aldehydes are reactive not only toward nucleic acids but also to many amino acids, and these aldehyde-protein interactions have been suspected of affecting many cellular functions, including DNA repair. To test this possibility we determined the effect of malondialdehyde (MDA), one of the most abundant intracellular aldehyde, on ultraviolet (UV) light- and benzo(a)pyrene diol epoxide (BPDE)-induced cytotoxicity and mutagenesis in human cells. We found that MDA treatment greatly sensitized cells to both UV- and BPDE-induced cell killing and that, MDA pre-treatment significantly enhanced UV-induced mutagenesis. Using in vitro DNA repair synthesis and host cell reactivation assays we found that MDA treatment of cells greatly inhibited nucleotide excision repair for both and UV light- and BPDE-induced DNA damage. Further experiments raise the possibility that the inhibitory effect on nucleotide excision repair is mainly caused by the direct interaction of MDA with cellular repair proteins. Together these results strongly suggest that intracellular aldehydes play an important role in oxidative stress-related mutagenesis and carcinogenesis through their inhibitory effect on DNA repair mechanisms as well as on induction of DNA damage.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2006

In vivo oxidative metabolism of a major peroxidation-derived DNA adduct, M1dG.

3-(2-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is a DNA adduct arising from the reaction of 2-deoxyguanosine with the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacteria and mammalian cells and is present in the genomic DNA of healthy human beings. It is also detectable, albeit at low levels, in the urine of healthy individuals, which may make it a useful biomarker of DNA damage linked to oxidative stress. We investigated the possibility that the low urinary levels of M1dG reflect metabolic conversion to derivatives. M1dG was rapidly removed from plasma (t(1/2) = 10 min) after i.v. administration to rats. A single urinary metabolite was detected that was identified as 6-oxo-M1dG by MS, NMR spectroscopy, and independent chemical synthesis. 6-Oxo-M1dG was generated in vitro by incubation of M1dG with rat liver cytosols, and studies with inhibitors suggested that xanthine oxidase and aldehyde oxidase are involved in the oxidative metabolism. M1dG also was metabolized by three separate human liver cytosol preparations, indicating 6-oxo-M1dG is a likely metabolite in humans. This represents a report of the oxidative metabolism of an endogenous DNA adduct and raises the possibility that other endogenous DNA adducts are metabolized by oxidative pathways. 6-Oxo-M1dG may be a useful biomarker of endogenous DNA damage associated with inflammation, oxidative stress, and certain types of cancer chemotherapy.

Animals↗
PubMed2006

COX-2 inhibitors modulate IL-12 signaling through JAK-STAT pathway leading to Th1 response in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease model of multiple sclerosis (MS). IL-12 plays a crucial role in the pathogenesis of EAE/MS and inhibition of IL-12 production or IL-12 signaling was effective in preventing EAE. Cyclooxygenase (COX-2) is a key enzyme promoting inflammation in rheumatoid arthritis and tumor induced angiogenesis. Recent studies have shown that COX-2 inhibitors prevent EAE, however, their mechanism of action is not fully understood. In this study, we show that in vivo treatment (i.p.) with 100 mug COX-2 selective inhibitors (LM01, LM08, LM11, and NS398), on every other day from day 0 to 30, significantly reduced the incidence and severity of EAE in SJL/J and C57BL/6 mice. Further analyses showed that the COX-2 inhibitors reduced neural antigen-induced IL-12 production, T cell proliferation and Th1 differentiation ex vivo and in vitro. The COX-2 inhibitors also decreased IL-12-induced T cell responses through blocking tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 proteins in T cells. These results demonstrate that COX-2 inhibitors ameliorate EAE in association with the modulation of IL-12 signaling through JAK-STAT pathway leading to Th1 differentiation and suggest their use in the treatment of MS and other Th1 cell-mediated autoimmune diseases.

Animals↗
PubMed2006

Site-specific synthesis of oligonucleotides containing malondialdehyde adducts of deoxyguanosine and deoxyadenosine via a postsynthetic modification strategy.

Malondialdehyde (MDA) and its reactive equivalent, base propenal, are products of oxidative damage to lipids and DNA, respectively; they are mutagenic in bacterial and mammalian systems, and MDA is carcinogenic in rats. MDA adducts of deoxyguanosine (M1dG), deoxyadenosine (OPdA), and deoxycytidine (OPdC) have been characterized. We have developed site-specific syntheses of M1dG and OPdA adducted oligonucleotides that rely on a postsynthetic modification strategy. This work provides an alternative route to the M1dG adducted oligonucleotide and, to date, the only viable strategy for the site-specific synthesis of OPdA-modified oligonucleotides. The stability of the modified oligonucleotides was examined by UV thermal melting studies (Tm). In contrast to the M1dG adduct, OPdA caused very little change in the Tm.

Deoxyadenosines↗
PubMed2006

Characterization of the amino acid adducts of the enedial derivative of teucrin A.

The toxicity of germander, a herb used to treat obesity, is attributed to cytochrome P450 activation of the furan ring of its major diterpenoid component (teucrin A) into a reactive metabolite capable of adducting proteins. 1,4-Enedials have been proposed to be the reactive products of metabolism, possibly arising from a rearrangement of putative epoxide intermediates. We synthesized the enedial derivative of teucrin A as well as the enedial derived from a model furan, 3-(4-methoxy-benzyloxymethyl)-furan, by dimethyldioxirane oxidation and characterized the products of their reactions with amino acids and peptides. The reactions of the model enedial, 2-(4-methoxy-benzyloxymethyl)-but-2-enedial, with N-acetyl lysine (NAL) afforded regioisomeric N-alkyl-3-pyrrolin-2-ones, differing in the substitution on the double bond of the heterocyclic ring. Novel products formed in the reactions of the model enedial with N-acetyl cysteine (NAC) and both NAC/NAL uncovered the existence of tautomerization between the enedial and a hydroxyenal, which was manifest by the loss of 4-methoxybenzylalcohol and the incorporation of a second molecule of NAC. The reactions of teucrin A-enedial with NAC and NAL afforded analogues of the products observed with the model enedial, and the existence of the tautomeric equilibrium resulted in epimerization of the proton (H12) adjacent to the former furan ring. This work further illuminates the complex chemical behavior of unsaturated dialdehydes as an important class of toxic metabolites and lays the foundation for studies of the protein targets of teucrin A-enedial.

Amino Acids↗
PubMed2006

Fatty acid amide hydrolase deficiency limits early pregnancy events.

Synchronized preimplantation embryo development and passage through the oviduct into the uterus are prerequisites for implantation, dysregulation of which often leads to pregnancy failure in women. Cannabinoid/endocannabinoid signaling via cannabinoid receptor CB1 is known to influence early pregnancy. Here we provide evidence that a critical balance between anandamide synthesis by N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and its degradation by fatty acid amide hydrolase (FAAH) in mouse embryos and oviducts creates locally an appropriate "anandamide tone" for normal development of embryos and their oviductal transport. FAAH inactivation yielding higher anandamide or experimentally induced higher cannabinoid [(-)-Delta9-tetrahydrocannabinol] levels constrain preimplantation embryo development with aberrant expression of Cdx2, Nanog, and Oct3/4, genes known to direct lineage specification. Defective oviductal embryo transport arising from aberrant endocannabinoid signaling also led to deferred on-time implantation and poor pregnancy outcome. Intercrossing between wild-type and Faah-/- mice rescued developmental defects, not oviductal transport, implying that embryonic and maternal FAAH plays differential roles in these processes. The results suggest that FAAH is a key metabolic gatekeeper, regulating on-site anandamide tone to direct preimplantation events that determine the fate of pregnancy. This study uncovers what we believe to be a novel regulation of preimplantation processes, which could be clinically relevant for fertility regulation in women.

Amidohydrolases↗
PubMed2005

Identification and functional characterization of brainstem cannabinoid CB2 receptors.

The presence and function of CB2 receptors in central nervous system (CNS) neurons are controversial. We report the expression of CB2 receptor messenger RNA and protein localization on brainstem neurons. These functional CB2 receptors in the brainstem were activated by a CB2 receptor agonist, 2-arachidonoylglycerol, and by elevated endogenous levels of endocannabinoids, which also act at CB1 receptors. CB2 receptors represent an alternative site of action of endocannabinoids that opens the possibility of nonpsychotropic therapeutic interventions using enhanced endocannabinoid levels in localized brain areas.

Animals↗
PubMed2005

Proceedings of a workshop on DNA adducts: biological significance and applications to risk assessment Washington, DC, April 13-14, 2004.

In April 2004, the Health and Environmental Sciences Institute, a branch of the International Life Sciences Institute, with support from the National Institute of Environmental Health Sciences, organized a workshop to discuss the biological significance of DNA adducts. Workshop speakers and attendees included leading international experts from government, academia, and industry in the field of adduct detection and interpretation. The workshop initially examined the relationship between measured adduct levels in the context of exposure and dose. This was followed by a discussion on the complex response of cells to deal with genotoxic insult in complex, interconnected, and interdependent repair pathways. One of the major objectives of the workshop was to address the recurring question about the mechanistic and toxicological relevance of low-concentration measured adducts and the presentations in the session entitled "Can low levels of DNA adducts predict adverse outcomes?" served as catalysts for further discussions on this subject during the course of the workshop. Speakers representing the regulatory community and industry reviewed the value, current practices, and limitations of utilizing DNA adduct data in risk assessment and addressed a number of practical questions pertaining to these issues. While no consensus statement emerged on the biological significance of low levels of DNA adducts, the workshop concluded by identifying the need for more experimental data to address this important question. One of the recommendations stemming from this workshop was the need to develop an interim "decision-logic" or framework to guide the integration of DNA adduct data in the risk assessment process. HESI has recently formed a subcommittee consisting of experts in the field and other key stakeholders to address this recommendation as well as to identify specific research projects that could help advance the understanding of the biological significance of low levels of DNA adducts.

Animals↗
PubMed2005

Indolyl esters and amides related to indomethacin are selective COX-2 inhibitors.

Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the nonsteroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides displayed time-dependent COX-2 inhibition with IC50 values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.

Amides↗
PubMed2005

Structural and functional differences between cyclooxygenases: fatty acid oxygenases with a critical role in cell signaling.

Cyclooxygenase (COX) catalyzes the first two steps in the conversion of arachidonic acid (AA) to prostaglandins (PGs). The reaction mechanism is well-defined and supported by extensive structural data. There are two isoforms of COX, which are nearly indistinguishable in structure and mechanism, however, COX-2 oxygenates neutral derivatives of AA that are poor substrates for COX-1. The best neutral substrate is 2-arachidonylglycerol, oxygenation of which produces an array of prostaglandin glyceryl esters (PG-Gs) that is nearly as diverse as the PGs. The mobilization of Ca2+ by subnanomolar concentrations of PGE2-G in RAW264.7 cells suggests the existence of a distinct receptor, and the formation of PG-Gs by zymosan-stimulated macrophages indicates that these species may be formed in vivo. These findings suggest that PG-Gs comprise a new class of lipid mediators, and that oxygenation of neutral derivatives of AA is a distinct function for the COX-2 isoform.

Animals↗
PubMed2005

Simultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry.

A high-performance liquid chromatography (HPLC) positive-ion electrospray ionization tandem mass spectrometry method for the quantification of prostaglandin glyceryl esters (PG-Gs), a newly discovered class of eicosanoids, is described. All four PG-Gs (PGE(2)-G, PGD(2)-G, PGF(2alpha)-G, and 6-keto-PGF(1alpha)-G) and the prostaglandins (PGs) that are formed by their hydrolysis are simultaneously quantified. Analytes were purified via reverse-phase solid-phase extraction, separated by reverse-phase HPLC, and quantified on a triple-quadrupole mass spectrometer using selected reaction monitoring. Quantification was achieved by stable isotope dilution employing penta-deuterated (PG-Gs) or tetra-deuterated (PGs) analogs. Analyte recovery from cell culture medium was >43% for all analytes at four different concentration levels. The limit of quantification is in the range of 25fmol on-column for each analyte and the analytes exhibit a linear response over approximately a 500-fold range. This method allows simultaneous profiling of several PG-Gs and PGs without multistep sample purification or derivatization.

Cells, Cultured↗
PubMed2005

Inhibition of cyclooxygenase with indomethacin phenethylamide reduces atherosclerosis in apoE-null mice.

Non-selective inhibition of cyclooxygenase (COX) has been reported to reduce atherosclerosis in both rabbit and murine models. In contrast, selective inhibition of COX-2 has been shown to suppress early atherosclerosis in LDL-receptor null mice but not more advanced lesions in apoE deficient (apoE(-/-)) mice. We investigated the efficacy of the novel COX inhibitor indomethacin phenethylamide (INDO-PA) on the development of different stages of atherosclerotic lesion formation in female apoE(-/-) mice. INDO-PA, which is highly selective for COX-2 in vitro, reduced platelet thromboxane production by 61% in vivo, indicating partial inhibition of COX-1 in vivo. Treatment of female apoE(-/-) mice with 5mg/kg INDO-PA significantly reduced early to intermediate aortic atherosclerotic lesion formation (44 and 53%, respectively) in both the aortic sinus and aorta en face compared to controls. Interestingly, there was no difference in the extent of atherosclerosis in the proximal aorta in apoE(-/-) mice treated from 11 to 21 weeks of age with INDO-PA, yet there was a striking (76%) reduction in lesion size by en face analysis in these mice. These studies demonstrate the ability of non-selective COX inhibition with INDO-PA to reduce early to intermediate atherosclerotic lesion formation in apoE(-/-) mice, supporting a role for anti-inflammatory approaches in the prevention of atherosclerosis.

Animals↗