Gene expression in human peripheral blood mononuclear cells upon acute ischemic stroke.

BACKGROUND AND PURPOSE: Ischemic stroke provokes a systemic inflammatory response. The purpose of this study was to characterize this response on the gene expression level in circulating mononuclear leukocytes from acute ischemic stroke (AIS) patients. METHODS: RNA from peripheral blood mononuclear cells (PBMCs) of AIS patients (24 + 2 hours after onset of symptoms) was analyzed with Affymetrix U133A GeneChips using a pooled design. We compared the gene expression signature from AIS patients (n = 20), stroke survivors (n = 15), patients with acute traumatic brain injury (ATBI, n = 15) and healthy control subjects without vascular risk factors (n = 15). RESULTS: Expression levels of 9682 probe sets with present calls on each GeneChip were compared. Between AIS patients and stroke survivors or between AIS patients and ATBI patients there were no significant differences in expression values of single genes after correction for multiple testing. However, comparison of the PBMC expression profiles from AIS patients and healthy subjects revealed significantly different expression (p = 0.012) of a single probe set, specific for phosphodiesterase 4 D (PDE4D). In order to detect modest expression differences in multiple genes with a presumed cumulative effect we studied the gene expression of functional groups of genes by global statistical tests. Analysis of 11 gene groups revealed differential expression between AIS patients and healthy subjects for genes involved in the inflammatory response (GeneOntology GO:0006954). Genes encoding the N-formyl peptide receptor-like 1 (FPRL1), interleukin-1 receptor antagonist (IL1RN) and complement component 3a receptor 1 (C3AR1) contributed most to the observed difference. CONCLUSIONS: This transcriptome analysis did not identify significant changes between circulating mononuclear cells from AIS patients 24 hours after stroke and closely matched stroke survivors. However, comparing AIS patients with healthy control subjects revealed measurable differences in PDE4D and in inflammatory response genes when considered as a set.

Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors.

The life span of normal fibroblasts in vitro (Hayflick limit) depends on donor age, and telomere shortening has been proposed as a potential mechanism. By quantitative fluorescence in situ hybridization and Southern blot analysis, we show progressive telomere loss to about 5 kb mean telomere restriction fragment length in fibroblasts from two adult donors within 40 population doublings, whereas in fibroblasts from two infant donors, telomere erosion is reduced, leaving a mean telomere restriction fragment length of approximately 7 kb at senescence (after approximately 60 population doublings). Aging of fibroblasts from both infant and adult donors was not accompanied by chromosomal abnormalities but was correlated with increased telomere repeat-binding factor 2 expression at both the protein and transcriptional level.

UV-induced DNA damage and mutations in Hupki (human p53 knock-in) mice recapitulate p53 hotspot alterations in sun-exposed human skin.

The major etiological agent contributing to human nonmelanoma skin cancer is sunlight. The p53 tumor suppressor gene is usually mutated in these tumors, and the mutations are "UV signature" single or tandem transitions at dipyrimidine sequences in the DNA-binding domain (DBD). Cells that harbor these characteristic mutations are already present in sun-exposed skin areas of healthy individuals, and small epidermal patches that are immunoreactive to anti-p53 antibody accrue as exposure increases. To explore carcinogen-specific human p53 mutation patterns experimentally, we generated a knock-in (Hupki) mouse in which the murine DBD of the p53 gene has been replaced by the homologous human p53 DBD segment; thus, the precise base sequence context frequently targeted by mutagens or endogenous mutagenic processes in human carcinogenesis is present in this strain (J. L. Luo et al., Oncogene, 20: 320-328, 2001). Here we show that when epidermal cells of Hupki mice (p53(ki/ki)) are irradiated in vivo with a single acute dose of UVB light, they accumulate UV photoproducts at the same locations of the p53 gene as human cells. Chronic exposure of Hupki mice (4.5 kJ/m(2) 5x/week for 4 weeks) results in the appearance of cell patches that stain intensely with the anti-p53 antiserum CM1. DNA preparations from 2 cm(2) sections of chronically irradiated Hupki epidermis harbor C to T and CC to TT mutations at two mutation hotspots identified in human skin cancer, one at codons 278-279, and one at codons 247-248; the latter is the most frequent UVB-associated mutation site in humans but not in p53 wild-type mice. Thus, Hupki keratinocytes with these p53 mutations encode an aberrant DBD identical in amino acid sequence to the mutant p53 molecules in human UV-induced tumors. The Hupki mouse model offers a new experimental tool in molecular epidemiology and biomedical research.

Knock-in mice with a chimeric human/murine p53 gene develop normally and show wild-type p53 responses to DNA damaging agents: a new biomedical research tool.

The high prevalence and great diversity of p53 tumor suppressor gene mutations in human tumors call for development of therapeutic molecules that rescue function of aberrant p53 protein. P53 mutations also offer new approaches to the study of the origins of mutations in human cancer. An experimental mouse model with a genetically modified but normal functioning p53 gene harboring the human rather than the murine core domain, would be of considerable benefit to research on both cancer therapeutics and etiology; however, it is uncertain whether such mice would permit biological functions of p53 to be retained. Using a Cre/lox P gene-targeting approach, we have constructed a human p53 knock-in (hupki) mouse strain in which exons 4-9 of the endogenous mouse p53 allele were replaced with the homologous, normal human p53 gene sequence. The chimeric p53 allele (p53(KI)) is properly spliced, transcribed in various tissues at levels equivalent to wild-type mice, and yields cDNA with the anticipated sequence, that is, with a core domain matching that of humans. The hupki p53 protein binds to p53 consensus sequences in gel mobility shift assays and accumulates in the nucleus of hupki fibroblasts in response to UV irradiation, as is characteristic of wild-type p53. Induction of various p53-regulated genes in spleen of gamma-irradiated homozygous hupki mice (p53(KI/KI)), and the kinetics of p53-dependent apoptosis in thymocytes are similar to results with wild-type (p53(+/+)) mice, further indicating normal p53 pathway function in the hupki strain. The mice are phenotypically normal and do not develop spontaneous tumors at an early age, in contrast to knock-out (p53(-/-)) strains with a defective p53 gene. The chimeric (p53(KI)) allele thus appears to provide a biological equivalent to the endogenous murine (p53(+)) gene. This strain is a unique tool for examining in vivo spontaneous and induced mutations in human p53 gene sequences for comparison with published human tumor p53 mutation spectra. In addition, the hupki strain paves the way for mouse models in pre-clinical testing of pharmaceuticals designed to modulate DNA-binding activity of human p53.

On the active principles of the spurge family (Euphorbiaceae). V. Extremely skin-irritant and moderately tumor-promoting diterpene esters from Euphorbia resinifera Berg.

The irritant and tumor-promoting principles were isolated from the latex of Euphorbia resinifera Berg. and from the resin derived from latex (euphorbium), which is commercially available as a drug. The irritant Euphorbia factors RL 5 (mixture), RL 6, RL 7, RL 8, and RL 10 were identified as tigliane-type 12-deoxyphorbol esters each bearing, in the 13 position, either long-chain, partially methyl-substituted acyl residues (10-16 carbon atoms) or short-chain acyl residues (4 or 5 carbon atoms) or a (substituted) phenylacetyl group with a 20-acetoxy group. Euphorbia factors RL 15 (mixture), RL 16, RL 17, RL 18, and RL 21 are the corresponding 20-deacetylated derivatives thereof. The irritant Euphorbia factors RL 11, RL 12, RL 22, RL 23 were characterized as esters of the tigliane type 12-deoxy-16-hydroxyphorbol, i.e., 13-0-phenylacetyl-16-0-benzoyl-12-deoxy16-hydroxyphorbol-20- acetate (RL 11) and 13, 16-0-phenylacetyl, tigloyl-12-deoxy-16-hydroxy-phorbol-20-acetate (RL 12), RL 22 and RL 23 representing the respective 20-deacetylated derivatives. A mixture of irritant factors, RL 13, was shown to represent long-chain 3-esters of ingenane-type ingenol with similar acyl residues (10-16 carbon atoms, partially methyl-substituted) to RL 5 (RL 15) above. A further group of E. resinifera factors was of the daphnane type: RL 9 was identified as the extremely irritant 9,13,14-orthophenylacetate of resiniferonol-20-(4-hydroxy-3-methoxy)phenylacetate (Resiniferatoxin), RL 14 as the corresponding 9,13,14-orthophenylacetate of resiniferonol, and RL 20 as 14-0-phenylacetylresiniferonol-20-(4-hydroxy-3-methoxy)-phenylacet ate (Proresiniferatoxin). The irritant factors specified below were accompanied by nonirritant esters of the tigliane type 12,20-dideoxyphorbol, i.e., RL 1 and RL 2, and of the lathyrane type ingol, i.e., RL 3 and RL 4. In tumor promotion experiments the mixture of homologous irritant factors RL 13 was equipotent with the standard tumor promoter TPA, but at 10 times the dose of TPA. Several others of the irritant factors had low activity as tumor promotors, but of the few tumors obtained in these experiments a high percentage was malignant. The very high irritant activity of the latex may be ascribed to resiniferatoxin (RL 9), representing a new class of rapidly acting skin irritants. No promoting activity was detected on administration of the highly irritant resiniferatoxin.

Induction of Epstein-Barr virus early antigens by tumor promoters of the diterpene ester type in Raji cells and specific (receptor) binding as compared to irritant and promoting activities.

Sixteen new diterpene esters (DTE) of the tigliane, ingenane, daphnane, and 1 alpha-alkyldaphnane types were investigated in two in vitro assays: as inhibitors of specific binding of 3H-labeled 12-O-tetradecanoylphorbol 13-acetate (TPA) to protein kinase C in a receptor preparation from mouse brain, and as inducers of Epstein-Barr virus (EBV) early antigens in Raji cells. Inhibition of binding of [3H]TPA to the receptor preparation by tigliane and ingenane DTE correlates with irritant activity in vivo, while some daphnane and 1 alpha-alkyldaphnane DTE inhibit binding of [3H]TPA in a less pronounced manner but still are very irritant. Tumor-promoting activity does not correlate consistently with the receptor-binding data. To test the hypothesis that early antigen induction in Raji cells by DTE is coupled to functional DTE receptors (protein kinase C), the latter were searched on these Raji cells by a 'cold acetone-filter assay' and shown to be present. The dependence of the early antigen induction rate on the concentration of the DTE tested was demonstrated. At a given concentration of DTE, differences in the induction rate between various DTE are seen. However, a clear quantitative correlation either between early antigen induction and receptor binding data in vitro, or early-antigen-inducing activity in vitro versus irritancy and tumor-promoting activity in vivo was not observed.

Toxicodynamics of tumour promoters of mouse skin. II. Binding to protein kinase C of some new diterpene esters and induction of luminol-enhanced chemoluminescence in mouse peritoneal neutrophils.

In binding competition assays using a protein kinase C preparation from mouse brain (particulate fraction) 3H-labelled 12-O-tetradecanoylphorbol-13-acetate (TPA), for a series of new diterpene esters (DTE) the relative binding affinity [rba = Kia(TPA)/Kia(DTE)] in relation to TPA was determined. A wide range of values was noticed, some of the DTE binding more strongly than TPA (rba greater than 1), others binding less strongly than TPA (rba less than 1) In comparative terms, competition for specific binding sites appears to correlate better with irritant than with promoting activity of the DTE. Using mouse peritoneal neutrophils, binding of [3H]-TPA was determined by a modification of the "cold-acetone filter assay"; saturation of high-affinity sites (Kda = 0.2 nM) was obtained at concentrations less than or equal to 1 nM, but there was also evidence for specific binding at "low-affinity" sites (Kda = 26 nM). Induction of chemoluminescence in the presence of luminol in mouse peritoneal neutrophils with a set of DTE usually elecited two peaks; at concentrations greater than or equal to 10 nM DTE a short-lived, "spike-like" response lasting only from 0 to about 5 min (phase A) its followed by a "plateau" response from about 5-120 min (phase B). This latter phase of chemoluminescence stimulation with luminol correlated well with the irritant potential of the DTE used. The sequence of the two phases can be inverted partially by using first TPA at 2,5 nM followed by a quick concentration increase to 100 nM; this indicates two different concentration-dependent events. As regards the intensity of the chemoluminescent response, quantitative but not qualitative differences between DTE were observed, which show some correlation with strong and weak tumour-promoting activity. Inhibition studies suggest the involvement of the myeloperoxidase/H2O2/Cl- system in the luminogenic response; it is suggested that the release of hypochlorite or a closely related oxidant may be instrumental in tumour promotion.

Toxicodynamics of tumour promoters of mouse skin. III. Specific binding of the tumour promoter thapsigargin as measured by the cold-acetone filter assay.

A method is described for measuring rapid, specific, and saturable binding of the skin irritant and tumour-promoting secretagogue thapsigargin (sesquiterpene lactone) to the microsomal fraction from mouse brain. Employing the tritium-labelled compound its apparent dissociation constant, Kd, and the maximal amount of binding Bmax are shown to be 9.8 nM and 1.9 pmol/mg protein respectively. Such a Kd for thapsigargin is similar to (a) its IC50 value for inhibiting Ca2+ uptake in the microsomal fraction from rat brain and (b) its EC50 values for inducing a rise in the cytoplasmic Ca2+ concentration of human platelets and histamine release from rat peritoneal mast cells. A positive correlation is found between the binding affinities of thapsigargin, thapsitranstagin, and trilobolide, their potencies as secretagogues and their lipophilicities. This correlation does not extend to the skin-irritant activities of the compounds thus emphasizing that their mechanism of action is unlike that of 12-O-tetradecanoylphorbol 13-acetate.

Biological assays for irritant tumor-initiating and -promoting activities. I. Kinetics of the irritant response in relation to the initiation-promoting activity of polyfunctional diterpenes representing tigliane and some daphnane types.

The kinetics of the irritant response on the mouse ear [erythema measured quantitatively by the irritant dose 50 (ID50)] and its relation to the initiation-promoting activity on the back skin of NMRI mice of some polyfunctional diterpene esters were investigated: (1) A very rapid and transient erythema response is associated with low or absent promoting activity (2) an early and more persistent erythema response in most cases is associated with moderate-to-high promoting activity, and (3) a relatively late onset but persistence up to at least 24 h of the erythema response is associated with high promoting activity. These results may indicate a relationship between the kinetics of the erythema response and the initiation-promoting activity of diterpene esters. The comparatively low promoting activity of esters carrying polyunsaturated acyl functions may indicate the importance of pharmacological parameters such as bioavailability and stability in the biological system, drug-receptor interaction, and "intrinsic activity" of the diterpene esters.

New approaches to understanding p53 gene tumor mutation spectra.

The first p53 gene mutation arising in a human tumor was described a decade ago by Baker et al. [S.J. Baker, E.R. Fearon, J.M. Nigro, S.R. Hamilton, A.C. Preisinger, J.M. Jessup, P. van Tuinen, D.H. Ledbetter, D.F. Barker, Y. Nakamura, R. White, B. Vogelstein, Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas, Science 244 (1989) 217-221]. There are now over 10,000 mutations extracted from the published literature in the IARC database of human p53 tumor mutations [P. Hainaut, T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores, M. Hollstein, C.C. Harris, R. Montesano, IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools, Nucleic Acids Res. 26 (1998) 205-213; Version R3, January 1999]. A large and diverse collection of tumor mutations in cancer patients provides important information on the nature of environmental factors or biological processes that are important causes of human gene mutation, since xenobiotic mutagens as well as endogenous mechanisms of genetic change produce characteristic types of patterns in target DNA [J.H. Miller, Mutational specificity in bacteria, Annu. Rev. Genet. 17 (1983) 215-238; T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709-715; S.P. Hussain, C.C. Harris, Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes, Cancer Res. 58 (1998) 4023-4037; P. Hainaut, M. Hollstein, p53 and human cancer: the first ten thousand mutations, Adv. Cancer Res. 2000]. P53 gene mutations in cancers can be compared to point mutation spectra at the HPRT locus of human lymphocytes from patients or healthy individuals with known exposure histories, and accumulated data indicate that mutation patterns at the two loci share certain general features. Hypotheses regarding specific cancer risk factors can be tested by comparing p53 tumor mutations typical of a defined patient group against mutations generated experimentally in rodents or in prokaryotic and eukaryotic cells in vitro. Refinements of this approach to hypothesis testing are being explored that employ human p53 sequences introduced artificially into experimental organisms used in laboratory mutagenesis assays. P53-specific laboratory models, combined with DNA microchips designed for high through-put mutation screening promise to unmask information currently hidden in the compilation of human tumor p53 mutations.

Microarray analysis of newly synthesized RNA in cells and animals.

Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

2-Chloroacetaldehyde induces epsilondA DNA adducts in DNA of Raji cells as demonstrated by an improved HPLC-fluorimetry method.

Etheno adducts in DNA are promutagenic lesions and are formed from vinyl chloride, urethane, and lipid peroxidation-derived products such as 4-hydroxynonenal. Such adducts were induced in a B-lymphoid cell line (Raji) by a metabolite of vinyl chloride, 2-chloroacetaldehyde. By modification of a method of Bedell et al., etheno dA was determined in 20 microg DNA by HPLC-fluorimetry. Our method has a detection limit of 5 fmol which is one two-hundredth that of the original method. By use of this method initial evidence was found that cigarette smoke can also induce etheno dA adducts in Raji cell DNA.

Specific binding of the tumor promoter TPA in various mouse organs as measured by a "cold acetone-filter assay'.

Rapid, specific, saturable and partially reversible binding of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) to a particulate fraction of mouse brain can be demonstrated by means of a "cold acetone-filter assay'; by washing with cold acetone at -20 degree C, nonspecific binding of the highly lipophilic [3H]TPA to membranes can be reduced to approximately 10% of the total binding. A comparative study of homogenates of several organs with this test revealed specific [3H]TPA binding/microgram DNA in the order brain much greater than lung approximately equal to spleen approximately equal to liver approximately equal to kidney approximately equal to thymus approximately equal to ovaries. Specific binding sites were also detected in 600 x g supernatant fractions of homogenates from epidermis, forestomach, glandular stomach and small intestine. Competition experiments showed displacement of [3H]TPA by TPA greater than phorbol-12,13-didecanoate greater than 12-deoxyphorbol-13-tetradecanoate greater than phorbol-12,13-dibutyrate (PDBu) greater than phorbol-12,13-diacetate greater than 4-O-methyl-TPA greater than 4 alpha-phorbol-12,13-didecanoate in the brain particulate fraction. Specific [3H]TPA binding is sensitive to heat, periodate and dithiothreitol, but is relatively insensitive to urea or to 1-5% solutions of several common detergents. It is estimated that the present binding test is approximately 500 times more sensitive than the widely-accepted [3H]PDBu assay; perhaps more important, the present method employs TPA, which is extremely effective both as a tumor promoter in vivo and as a pleiotropic effector of cells in vivo and in vitro.

Specific lipid components in membrane receptors of 12-O-tetradecanoylphorbol-13-acetate as revealed by direct photoaffinity labelling.

The use of [20-3H]-12-O-tetradecanoylphorbol-13-acetate (3H-TPA) as a direct photoaffinity probe of receptors was investigated. When membrane receptors in a particulate fraction from mouse brain were loaded with the high-affinity agonist 3H-TPA, irradiation of such preparations with ultraviolet (UV) light resulted in specific irreversible binding of the label to the membrane lipids phosphatidylethanolamine (PE) and phosphatidylserine (PS); evidence for labelling of proteins was lacking. The labelled lipids were tentatively identified by co-chromatography; they corresponded to selected reference lipids obtained by photoaffinity labelling. When a variety of natural and synthetic reference lipids was irradiated in the presence of 3H-TPA, photoadducts were obtained primarily from natural lipids containing unsaturated acyl chains in position 2. The synthetic 1,2-dipalmitoyl derivatives of phosphatidylcholine (PC), PE and phosphatidyl glycerol were refractory. When the photoadducts from PC and PE were treated with phospholipases A2 and C, the chromatographic pattern of cleavage products obtained was in accordance with that of phospholipids carrying the 3H-TPA label on the acyl moiety in the 2-position. Since previous evidence had suggested that a lipid-protein complex was the phorbol ester receptor, it may, therefore, be concluded that the receptor(s) contain PE and PS in specific interaction with protein(s). Accordingly, PE- and PS-dependent enzymes (or other proteins) are considered prime candidates for phorbol ester receptors.

Epstein-Barr virus episomes as targets for cigarette smoke- and gamma-irradiation-induced DNA damage: studies on the EBNA-1 region by a new gene-specific technique.

Following our demonstration of cytochrome P450-independent DNA damage induced by aqueous solutions of cigarette smoke in human mucosal cells in vivo, and in a lymphoblastoid cell line, we have developed a new technique to demonstrate gene-region specific DNA damage, with the EBNA-1 gene present in multiple nuclear matrix-attached episomes in Raji cells serving as an amplified target. DNA was extracted from Raji cells treated by gamma-irradiation or aqueous solutions of cigarette smoke; adducted bases or other damage were removed chemically by depurination/alkali treatment. Single-strand breaks induced directly by cigarette smoke as well as DNA cleaved at the site of former adducts were end-labelled either with alpha-[32P]dCTP or with biotin-16-dUTP. With 32P-labelling, a dose-dependent increase in DNA labelling was seen for different concentrations of cigarette smoke; undiluted smoke produced a similar amount of damage as 22.4 Gy of gamma-irradiation. For isolation of DNA regions that contained biotin label at the sites of former damage, DNA was cut by restriction endonucleases and 3-kb-fragments including the target gene, EBV-EBNA-1, were isolated by agarose-gel electrophoresis. Those containing biotin were selected on streptavidin-coated magnetic beads. PCR amplification of the bound DNA revealed EBNA-1 DNA only when cells were pretreated with either cigarette smoke or gamma-irradiation. The presented method thus provides a new approach for detecting gene-specific damage in a readily accessible target, EBV episomes. The method is also potentially applicable for studying single-copy genes such as p53, the types of adducts involved, and quantitative aspects of DNA damage and its repair.

Structure-activity relations of polyfunctional diterpenes of the daphnane type. I. Revised structure for resiniferatoxin and structure-activity relations of resiniferonol and some of its esters.

Base catalyzed transesterifications of resiniferatoxin afforded the 9, 13, 14-orthophenylacetate (III), as well as (4-hydroxy-3-methyoxyphenyl) acetic acid methyl ester (II). Reesterification of III with homovanillic acid showed that the previously proposed structure for resiniferatoxin has to be revised to resiniferonol-9, 13, 14-orthophenylacetate-20-(4-hydroxy-3-methoxyphenyl) acetate (I). From III, different 20 esters (I, IV-IX) were prepared. The orthophenylacetate III was cleaved by Acidic hydrolysis to yield the 14-phenylacetate X. Subsequent alkaline transesterifications of X afforded the parent alcohol resiniferonol (XI), Starting from XI, the 14, 20-diesters XII and XV and the 9, 13, 20-diesters xii and XV and the 9, 13, 14-orthoesters XIII, XIV, XVI, XVII and XVIII were obtained. All esters were tested for irritant activity on the mouse ear. Some aspects of structure-activity relations of irritancy of resiniferonol esters are established and discussed.

Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji.

Human lymphoid cells (Raji) were exposed to water-soluble compounds from cigarette smoke (CS) generated in a smoking machine. DNA damage, as detected by alkaline single-cell microelectrophoresis (COMET assay), was induced in a time- and concentration-dependent manner in the cells. Most of the rapidly induced DNA damage was attributable to direct-acting compounds since cytochrome P450-related metabolic activities (ethoxy- and pentoxyresorufin-O-deethylases and coumarin-7-hydroxylase) were absent or very low. In addition, induction of DNA damage could be inhibited only slightly by beta-naphthoflavone and coumarin. Vitamin C enhanced DNA damage in Raji cells probably by redox cycling of catechol and hydroquinone present in CS implicating reactive oxygen intermediates as another source of DNA damage. N-acetylcysteine, a radical scavenger and glutathione precursor, reduced DNA damage in Raji cells when exposure to CS was followed by 2 h post-incubation in culture medium. Unrepaired DNA damage caused by CS persisted longer than gamma-irradiation-induced DNA damage. Among the CS constituents, acrolein, but not formaldehyde and acetaldehyde, induced DNA damage although less intensely than CS itself. At 50 and 100 microM concentrations, acrolein also inhibited repair of gamma- irradiation-induced DNA damage in the COMET assay. Inhibition of DNA synthesis by acrolein at 50 microM was demonstrated by an immunochemical assay for bromo-deoxyuridine incorporation; however, inhibition of a representative repair enzyme, 8-oxoguanosine hydrolase, by either CS or acrolein was not observed. The present results further confirm the presence of direct-acting genotoxic components and inhibitors of DNA repair in the gas phase of tobacco smoke, that may contribute to DNA damage and smoking-associated cancers of the upper aerodigestive tract.

Validation of two test systems for detecting tumor promoters and EBV inducers: comparative responses of several agents in DR-CAT Raji cells and in human granulocytes.

The response to a number of agents has been compared in two short-term assays used for the detection of virus inducers and tumor promoters: (i) induction of the EBV-DR-promoter in Raji cells, as measured by DR-CAT induction (DR-CAT test) and (ii) induction of the oxidative burst in human PMN, as measured by chemiluminescence in the presence of luminol or lucigenin (CL test). In order to validate the two assays, we have investigated the responses to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1,2-dioctanoylglycerol (DAG), phospholipase C (PLC EC-3-1-4-30) and ionophore A23187, which are active in both systems: arachidonic acid, linoleic acid and NaCl were found active only in the CL test. Staurosporine (protein kinase inhibitor), tamoxifen (estrogen antagonist and protein kinase C inhibitor), forskolin (protein kinase A activator), R59949 (diacylglycerol kinase inhibitor), curcumin and N-acetyl-L-cysteine (scavengers of reactive oxygen species) and NaCl acted as inhibitors. A good concordance of the EC50 values of inducing substances was found between the two assays, except for A23187 and DAG, which were required at much higher concentrations in the DR-CAT test. The inhibition patterns by the panel of inhibitors revealed similarities and discrepancies in the induction pathways between the two systems, providing information on their mode of action. The two assays, which complement each other, were shown to detect a number of known or suspected EBV inducers or tumor promoters, and thus appear useful for screening of new compounds or mixtures as well as of potential antiviral and antipromoting substances.

Effect of ethionine on the rough endoplasmic reticulum from male and female rat liver.

Ethionine causes a decrease in the amount of rough endoplasmic reticulum in rat liver, the effect being greater in female than in male rats. Rough endoplasmic reticulum isolated from rat liver 24 hr after ethionine injection and stripped of its ribosomes partially lost its in vitro ribosome binding capacity. However, no differences were detected between the binding affinities of ribosomes, isolated from either untreated animals or intoxicated rats, to stripped rough membranes derived from normal rats. Structural changes occur in the rough endoplasmic reticulum of the ethionine treated rats, while the ribosomes are still bound to the membrane.