Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Mitochondrial DNA in human malignancy.

Alterations in expression of mitochondrial DNA (mtDNA)-encoded polypeptides required for oxidative phosphorylation and cellular ATP generation may be a general characteristic of cancer cells. Mitochondrial DNA has been proposed to be involved in carcinogenesis because of high susceptibility to mutations and limited repair mechanisms in comparison to nuclear DNA. Since mtDNA lacks introns, it has been suggested that most mutations will occur in coding sequences and subsequent accumulation of mutations may lead to tumor formation. The mitochondrial genome is dependent upon the nuclear genome for transcription, translation, replication and repair, but precise mechanisms for how the two genomes interact and integrate with each other are poorly understood. In solid tumors, elevated expression of mtDNA-encoded subunits of the mitochondrial electron respiratory chain may reflect mitochondrial adaptation to perturbations in cellular energy requirements. In this paper, we review mitochondrial genomic aberrations reported in solid tumors of the breast, colon, stomach, liver, kidney, bladder, head/neck and lung as well as for hematologic diseases such as leukemia, myelodysplastic syndrome and lymphoma. We include data for elevated expression of mtDNA-encoded electron respiratory chain subunits in breast, colon and liver cancers and also the mutations reported in cancers of the colon, stomach, bladder, head/neck and lung. Finally, we examine the role of reactive oxygen species (ROS) generated by mitochondria in the process of carcinogenesis.

DNA methylation and the association between genetic and epigenetic changes: relation to carcinogenesis.

This paper examines the relationship between DNA mutagenic lesions, DNA methylation and the involvement of these changes in the process of carcinogenesis. Many types of DNA damage (oxidative lesions, alkylation of bases, abasic sites, photodimers, etc.) interfere with the ability of mammalian cell DNA to be methylated at CpG dinucleotides by DNA-methyltransferases (DNA-MTases). This can result in altered patterns in the distribution of 5-methylcytosine (5MeC) residues at CpG sites. Methylation of DNA is an epigenetic change that by definition is heritable, can result in changes in chromatin structure, and is often accompanied by modified patterns of gene expression. The presence of 5MeC in DNA, as well as oxidative stress induced by the free radical nitric oxide, can interefere with the repair of alkylation damage, thereby increasing the level of potentially mutagenic lesions. CpG sites in DNA represent mutational hotspots, with both the presence of 5MeC in DNA and the catalytic activity of DNA-MTases being intrinsically mutagenic. The process of carcinogenesis has frequently been associated with an increased expression of DNA-MTase activity, accompanied by either hypermethylation or hypomethylation of target cell (progenitor tumor cell) DNA. In addition, there is evidence that overexpression of DNA-MTase activity could result in increased cytosine methylation at non-CpG sites. A variety of chemicals can alter the extent of DNA methylation in mammalian cells. These include inhibitors of topoisomerase II, as well as inhibitors of DNA synthesis, microtubule formation, histone deacetylation, transmethylation, etc. Genetic and epigenetic changes in DNA have a profound influence on one another and could play a major role in the process of carcinogenesis, by modulating both the extent and the pattern of gene expression.

The NIEHS Predictive-Toxicology Evaluation Project.

The Predictive-Toxicology Evaluation (PTE) project conducts collaborative experiments that subject the performance of predictive-toxicology (PT) methods to rigorous, objective evaluation in a uniquely informative manner. Sponsored by the National Institute of Environmental Health Sciences, it takes advantage of the ongoing testing conducted by the U.S. National Toxicology Program (NTP) to estimate the true error of models that have been applied to make prospective predictions on previously untested, noncongeneric-chemical substances. The PTE project first identifies a group of standardized NTP chemical bioassays either scheduled to be conducted or are ongoing, but not yet complete. The project then announces and advertises the evaluation experiment, disseminates information about the chemical bioassays, and encourages researchers from a wide variety of disciplines to publish their predictions in peer-reviewed journals, using whatever approaches and methods they feel are best. A collection of such papers is published in this Environmental Health Perspectives Supplement, providing readers the opportunity to compare and contrast PT approaches and models, within the context of their prospective application to an actual-use situation. This introduction to this collection of papers on predictive toxicology summarizes the predictions made and the final results obtained for the 44 chemical carcinogenesis bioassays of the first PTE experiment (PTE-1) and presents information that identifies the 30 chemical carcinogenesis bioassays of PTE-2, along with a table of prediction sets that have been published to date. It also provides background about the origin and goals of the PTE project, outlines the special challenge associated with estimating the true error of models that aspire to predict open-system behavior, and summarizes what has been learned to date.

The beneficial effects of dietary restriction: reduced oxidative damage and enhanced apoptosis.

There is compelling evidence for the central role of oxidative damage in the aging process and for the participation of reactive oxygen species in tumor initiation and promotion. Caloric restriction (CR) or energy restriction retards age-associated increases in mitochondrial free-radical production and reduces the accumulation of oxidatively damaged cell components. CR has also been shown to slow down age-related declines in various repair capabilities, including some types of DNA repair. It is proposed that inhibitors of mitochondrial electron transport and/or uncouplers of oxidative phosphorylation (rotenone, amytal, amiodarone, valinomycin, etc.), when used at extremely low doses, could mimic the effects of CR in model systems. The objective is to lower mitochondrial free-radical production by decreasing the fraction of electron carriers in the reduced state. In addition to a variety of other effects, CR has been shown to increase the rate of apoptosis, particularly in preneoplastic cells, and in general, to promote elevated levels of free glucocorticoids (GCs). GCs are known to induce tissue-specific apoptosis and to upregulate gap-junction-mediated intercellular communication (GJIC). Tumor promoters like phorbol esters have the opposite effect, in that they inhibit both the process of apoptosis and GJIC. The enzyme poly (ADP-ribose) polymerase (PARP) is thought to play a central role in apoptosis, in a manner that has been highly conserved in evolution. There is good evidence that the apoptosis-associated Ca/Mg-dependent DNA endonuclease is maintained in a latent form by being poly (ADP-ribosylated). Apoptosis would require the removal of this polymer from the endonuclease, and, most likely, its removal from topoisomerase II and histone H1 as well. The role of poly (ADP-ribose) in apoptosis, carcinogenesis, and aging could be studied by the use of modulators of PARP activity (3-aminobenzamide, 3-nitrosobenzamide, 1% ethanol, etc.), inhibitors of poly ADP-ribose) glycohydrolase activity (ethacridine, 43 degrees C, etc.), and inhibitors of the PARP-specific protease (interleukin-1 beta converting enzyme (ICE)-like protease). Also, it would be of interest to determine if CR can decrease the half-life of poly (ADP-ribose), upregulate GJIC, and modulate the activities of PARP, the glycohydrolase, and the PARP-specific protease, factors potentially important in these processes.

Hydrogen peroxide stimulates rat colonic prostaglandin production and alters electrolyte transport.

The changes in short circuit current (electrogenic Cl- secretion) of rat colon brought about by xanthine/xanthine oxidase in the Ussing chamber were inhibited by catalase and diethyldithiocarbamate, but not by superoxide dismutase. These results, the reproduction of the response with glucose/glucose oxidase and with exogenous H2O2, and the lack of effect of preincubation with deferoxamine or thiourea implicate H2O2, and not O2- or OH., as the important reactive oxygen metabolite altering intestinal electrolyte transport. 1 mM H2O2 stimulated colonic PGE2 and PGI2 production 8- and 15-fold, respectively, inhibited neutral NaCl absorption, and stimulated biphasic electrogenic Cl secretion with little effect on enterocyte lactic dehydrogenase release, epithelial conductance, or histology. Cl- secretion was reduced by cyclooxygenase inhibition. Also, the Cl- secretion, but not the increase in prostaglandin production, was reduced by enteric nervous system blockade with tetrodotoxin, hexamethonium, or atropine. Thus, H2O2 appears to alter electrolyte transport by releasing prostaglandins that activate the enteric nervous system. The change in short circuit current in response to Iloprost, but not PGE2, was blocked by tetrodotoxin. Therefore, PGI2 may be the mediator of the H2O2 response. H2O2 produced in nontoxic concentrations in the inflamed gut could have significant physiologic effects on intestinal water and electrolyte transport.

Immune system control of rat and rabbit colonic electrolyte transport. Role of prostaglandins and enteric nervous system.

The role of the immune system in controlling intestinal electrolyte transport was studied in rat and rabbit colon in Ussing chambers. A phagocyte stimulus, the chemotactic peptide FMLP, and a mast cell stimulus, sheep anti-rat IgE, caused a brief (less than 10 min) increase in short-circuit current (Isc). Products of immune system activation, platelet-activating factor (PAF) and reactive oxygen species (ROS), caused a sustained, biphasic increase in the Isc. Ion replacement and flux studies indicated that these agonists stimulated electrogenic Cl secretion and inhibited neutral NaCl absorption; responses that were variably inhibited by the cyclooxygenase blockers indomethacin and piroxicam. Lesser degrees of inhibition by nordihydroguaiaretic acid could be accounted for by decreased prostaglandin synthesis rather than by lipoxygenase blockade. Tetrodotoxin, hexamethonium, and atropine also inhibited immune agonist-stimulated Isc, but had no effect on immune agonist-stimulated production of PGE2 or PGI2. These results indicate that immune system agonists alter intestinal epithelial electrolyte transport through release of cyclooxygenase products from cells in the lamina propria with at least 50% of the response being due to cyclooxygenase product activation of the enteric nervous system. The immune system, like the enteric nervous system and the endocrine system, may be a major regulating system for intestinal water and electrolyte transport in health and disease.

A new epistasis group for the repair of DNA damage in bacteriophage T4: replication repair.

The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication.

Characterization and partial purification of the plasminogen activator from human neuroblastoma cell line, SK-N-SH. A comparison with human urokinase.

The plasminogen activator liberated by cells of human neuroblastoma strain SK-N-SH was purified up to 400-fold, with a 40% recovery of activity, by a relatively simple procedure. This involved (NH4)2SO4 precipitation, followed by chromatography on both Affi-Gel Blue and p-aminobenzaminidine-Sepharose. The SK-N-SH activator was shown to differ from human urokinase with respect to immunological specificity, the molecular weights and isoelectric points of their enzymatically active species, the ability to be activated by fibrin and their relative sensitivities to inactivation by diisopropyl fluorophosphate. The average molecular weights of the enzymatically active species derived from strain SK-N-SH were shown to be 66,500, 64,500, 60,500 and 37,500. In the presence of fibrin, the SK-N-SH plasminogen activator appeared to be stimulated approximately 16-fold, with no apparent stimulation of urokinase activity. Urokinase is inactivated by diisopropyl fluorophosphate at a rate 6.4-fold faster than that of the SK-N-SH activator.

Increased neurite development and plasminogen activator expression by exposure of human neuroblastoma cells to plasminogen-deficient growth medium.

Growth of human neuroblastoma strain SK-N-SH in a plasminogen-deficient medium results in about a 40% increase in the number of differentiated cells (cells with a neurite-like process at least 50 micrometers in length) and about a five-fold increase in the amount of plasminogen activator liberated per cell. Plasminogen deficiency has no effect on the growth rate of SK-N-SH cells. These results are consistent with the hypothesis that plasminogen activator is involved in neuroblast development.

L cell DNA ligase joins RNA to DNA on a DNA template.

L cell DNA ligase catalyzes a covalent linkage between 5'-hosphoryl oligodeoxyribonucleotides and 3'-hydroxyl oligoribonucleotides on a complementary polydeoxyribonucleotide template. This reaction occurs to a substantially lesser extent than does the sealing of DNA to DNA. The joining of [5'32P]d(pA)12-18 to (Ap)11A on poly[d(T)] or of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) ON POLY[D(C)] was demonstrated by the formation of alkaline phosphatase resistant radioactivity. The 32P of the hybrid reaction products became sensitive to the action of alkaline phosphatase after treatment with alkali. Furthermore, hydrolysis of the products of the linkage of [5'-32P]d(pG)12-18 to 5'-hydroxyl, 3'-hydroxyl oligo(I) on poly[d(C)] with micrococcal nuclease and spleen phosphodiesterase resulted in the formation of [3'-32P]IMP. Attempts to seal [5'-32p[-(pA)12 to d(Ap)11-17A on poly[d(T) or [5'-32P]oligo(pI) to d(Gp)11-17G on poly[d(C)] were unsuccessful.

Fibrinolytic activity associated with cultured human neoplastic and normal cells.

Fibrinolytic activity was studied in a number of different established as well as secondary human cell cultures derived from both malignant and normal tissues. The ability to degrade [25I]-labeled fibrin was found to be characteristic of some malignant cultures as well as some normal cultures, and to be dependent upon the presence of serum. For the most part, this activity was detected in cultures with a relatively short in vitro passage history (less than 30 passages). Low passaged colon and rectal carcinoma cells, HCT-8 and HRT-18, as well as normal rectal, colon and foreskin fibroblasts were positive for fibrinolytic activity, while long established (greater than 100 passages) cultures of malignant cells (colon carcinoma, HeLa, Hep-2, KB) as well as normal cells (HEI, AV3) were negative. It is proposed that although some normal cells synthesize plasminogen activators, the fibrinolytic capability of both malignant and normal cells may be lost on prolonged in vitro cultivation.

Evidence for four deoxynucleoside kinase activities in extracts of Lactobacillus leichmannii.

Extracts of Lactobacillus leichmannii (ATCC 7830) catalyze the phosphorylation of the four principal deoxynucleosides. Thymidine, deoxyguanosine, and deoxycytidine kinase activities were found to be optimal with deoxyadenosine triphosphate as the phosphoryl donor, whereas deoxycytidine triphosphate was the optimal donor for deoxyadenosine kinase activity. L. leichmannii catalyzes the conversion of deoxycytidine to deoxyuridylic acid, probably by a pathway involving deoxycytidylate deaminase.

Demonstration of a deoxycytidine kinase activity in extracts of Bacillus megaterium KM.

Extracts of Bacillus megaterium KM have been shown to possess a deoxycytidine kinase activity which functions optimally with guanosine triphosphate as the phosphoryl donor.

Properties of thymineless strains of Bacillus megaterium.

Both Bacillus megaterium KM:T(-)R(1), a strain partially resistant to thymineless death, and strain KM:T(-), the parent strain, can satisfy their thymine requirement with either thymidine, 5-methyldeoxycytidine, or 5-methyluridine. Neither strain can use 5-methylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, or 5-aminouracil for this purpose. Strain KM:T(-)R(1) requires as little as 0.01 mM thymine for maximum growth, whereas strain KM:T(-) requires 0.10 to 0.20 mM thymine. Lysogenic KM:T(-)R(1) dies more rapidly in the presence of mitomycin C than the corresponding phage-sensitive strain. Unexpectedly, the lysogenic strain was found to be less sensitive to thymineless death than the phage-sensitive strain. Lysogenic KM:T(-)R(1) is induced by exposure to mitomycin C and by thymineless incubation. It is concluded that thymineless death occurs by a mechanism which is unrelated to phage induction and that a major lethal effect of mitomycin C is probably a consequence of phage induction.