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Biomedical subjects

G N Wogan

Publications and source records attributed to G N Wogan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Nitric oxide production in SJL mice bearing the RcsX lymphoma: a model for in vivo toxicological evaluation of NO.

SJL mice spontaneously develop pre-B-cell lymphoma that we hypothesized might stimulate macrophages to produce nitric oxide (NO.). Transplantation of an aggressive lymphoma (RcsX) was used to induce tumor formation. Urinary nitrate excretion was measured as an index of NO. production and was found to increase 50-fold by 13 days after tumor injection. NO. production was prevented by the addition of a nitric oxide synthase (NOS) inhibitor. The expression of inducible NOS (iNOS) in various tissues was estimated by Western blot analysis and localized by immunohistochemistry. The synthase was detected in the spleen, lymph nodes, and liver of treated but not control mice. To assess whether the iNOS-staining cells were macrophages, spleen sections from ResX-bearing animals were costained with anti-iNOS antibody and the anti-macrophage antibody moma-2. Expression of iNOS was found to be limited to a subset of the macrophage population. The concentration of gamma-interferon, a cytokine known to induce NO. production by macrophages, in the serum of tumor-bearing mice, was measured and found to be elevated 25-fold above untreated mice. The ability of ResX-activated macrophages to inhibit splenocyte growth in primary culture was estimated and macrophage-derived NO. was found to inhibit cell division 10-fold. Our findings demonstrate that ResX cells stimulate NO. production by macrophages in the spleen and lymph nodes of SJL mice, and we believe this experimental model will prove useful for study of the toxicological effects of NO. under physiological conditions.

Animals↗
PubMed1996

Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells.

Peroxynitrite is a powerful oxidant formed through reaction of nitric oxide with superoxide. Because activated macrophages can produce both nitric oxide and superoxide, it has been proposed that peroxynitrite may contribute to cytotoxicity and increased cancer risks associated with the inflammatory response during chronic infections. We therefore investigated mutagenicity of peroxynitrite in the supF gene of the pSP189 shuttle vector as a mutation target. The plasmid was exposed to 2.5 mM peroxynitrite in vitro, then replicated in Eschericia coli MBL50 and in human AD293 cells. Mutation frequency increased 21-fold in pSP189 replicated in E. coli and 9-fold in plasmid replicated in human cells. Mutations were clustered within the 5' region of the supF gene in plasmids replicated in bacteria. The hot spots were located at positions 108, 113, 116, 124, 126 and 141; more than 25% of all mutations occurred at position 124. Following replication in human cells, mutations were more widely distributed over the gene, with hot spots at positions 113, 124, 133, 156 and 164; 15% occurred at position 124. In both systems, the majority of mutations occurred at G:C base pairs, predominantly involving G:C-->T:A transversions (65% when replication was in bacteria and 63% when in human cells). G:C-->C:G transversions were observed at lower frequency (28% in MBL50 and 11% in AD293 cells), and 11% of mutations found in vectors replicated in AD293 cells were G:C-->A:T transitions. A greater number of large deletions, insertions, tandem and multiple mutations occurred in plasmid replicated in AD293 cells. Differences in mutation spectra following replication in the two systems may be attributable to differences in recognition and repair of the lesions and/or properties of the replication apparatus.

Base Sequence↗
PubMed1996

Percutaneous absorption and tissue distribution of [3H]diacetoxyscirpenol (anguidine) in rats and mice.

Acute toxicity, absorption, excretion, and tissue distribution of topically administered diacetoxyscirpenol (DAS, anguidine) were studied in Fischer rats and CD-1 mice. Mortality (75%) was observed in rats treated with a single dose of 2.625 mg of DAS to 1.44 cm2 of skin, whereas the proportionate (0.75 mg to 0.42 cm2 of skin) and even higher doses were not lethal to mice. Histopathological lesions induced in the rat were similar to those observed by administration through other routes, mainly involving lymphohematopoietic tissues and the gastrointestinal tract. Although lesions in internal organs were less severe, the skin of the mouse was more severely damaged at the application site than that of the rat. During the 90-min period after topical application of a single dose of [3H]DAS, the rat absorbed and retained more [3H]DAS and excreted less radioactivity through urine and feces than the mouse. By 24 hr after treatment, the rat had absorbed, excreted, and retained about twice as much [3H]DAS as had the mouse (p < 0.05 or < 0.005). At 7 days posttreatment, the rat had absorbed more than four times the amount of [3H]DAS than had the mouse (13.1 vs 57.5%; p < 0.005). However, tissues of the mouse retained a higher proportion of administered radioactivity (4.1%) than those of the rat (1.0%; p < 0.05). Total excretion of radiolabel by the rat was approximately sixfold higher than that of the mouse (56 to 9%; p < 0.005). The ratio of excretion in urine to that in feces in the rat was about 2 to 1 (37 to 18%) and in the mouse was about 3.5 to 1 (7 to 2%). Significant differences in the time course of tissue distribution of [3H]DAS in the rat and mouse were found when data were expressed as the percentage of absorbed dose present in tissues or as specific radioactivity (dpm) per gram tissue. These results demonstrated a significant interspecies difference in acute percutaneous toxicity of DAS and different patterns of absorption, excretion, and tissue distribution of topically administered [3H]DAS in rats and mice.

Absorption↗
PubMed1996

Use of carcinogen-DNA and carcinogen-protein adduct biomarkers for cohort selection and as modifiable end points in chemoprevention trials.

Clinical cancer prevention studies that use disease as an end point are of necessity large, lengthy and extremely costly. Development of the field of cancer chemoprevention is being accelerated by the application of intermediate markers to preclinical and clinical studies. One class of potentially useful biomarkers comes from studies of cancer risks derived from exposure to environmental carcinogens of both endogenous and exogenous origins. Sensitive and specific analytical methods have been developed for detecting and quantifying levels of covalent adducts of several important classes of carcinogens with cellular DNA and blood proteins at ambient levels of exposure. Such biomarkers can be applied to the preselection of exposed individuals for study cohorts, thereby reducing study-size requirements. Levels of these carcinogen-DNA and carcinogen-protein adducts can be modulated by some classes of chemopreventive agents. In this regard, these markers can also be used to rapidly assess the efficacy of preventive interventions such as exposure abatement and chemoprevention. However, the successful application of these biomarkers to prevention trials will be dependent upon prior determination of the associative or causal role of the marker to the carcinogenic process, establishment of the relationship between dose and response, and appreciation of the kinetics of adduct formation and removal.

Anticarcinogenic Agents↗
PubMed1995

Nitric oxide production in relation to spontaneous B-cell lymphoma and myositis in SJL mice.

SJL mice spontaneously develop B cell lymphomas (historically described as reticulum cell sarcomas) by 12 months of age and inflammatory muscle disease (myositis) by 6 months of age. Tumors originate in mesenteric lymph nodes and in Peyer's patches and resemble human germinal center lymphomas. The growth of reticulum cell sarcomas is completely dependent on cytokine production by normal T cells. The spontaneous myositis, which resembles human idiopathic myositis, is characterized by various abnormalities in skeletal muscle, including infiltration with inflammatory cells consisting primarily of macrophages. The participation of different cytokines in the pathogenesis of the lymphoma and the massive invasion of macrophages into muscle tissues led us to investigate the possible involvement of nitric oxide (NO.), which is known to be synthesized by activated macrophages under inflammatory conditions. Elevated NO. production, measured by urinary nitrate excretion, by SJL mice in comparison with BALB/c control mice was observed as early as 7 weeks of age. Both aging and degree of spontaneous myositis correlated with increased nitric oxide production. Oral administration of N-monomethyl-L-arginine, an inhibitor of nitric oxide synthase (NOS), reduced urinary nitrate excretion and also the severity of myositis. Immunohistochemical analysis revealed the presence of inducible NOS (iNOS) in cells in the spleen, lymph nodes, and skeletal muscle. The iNOS is primarily responsible for the enhanced nitric oxide production. Morphology of cells that stained positive for iNOS was similar to that of macrophages infiltrating into the affected tissues. Chronic production of elevated amounts of nitric oxide by the SJL mice, therefore, provides a useful in vivo model for future studies of cellular damage resulting from endogenously produced NO.in combination with oxygen radicals.

Amino Acid Oxidoreductases↗
PubMed1995

Tissue distribution of DNA adducts in pre-weanling BLU:Ha mice treated with a tumorigenic dose of fluoranthene.

Fluoranthene (FA), a ubiquitous atmospheric pollutant, is tumorigenic to the lung when injected into BLU:Ha mice 1, 8, and 15 days after birth. DNA adducts were measured by a modified HPLC-32P-postlabeling method in target (lung) and non-target (liver, kidney, spleen plus thymus) organs of 16-day-old mice 24 h after the last treatment with a tumorigenic dose (3.5 mg/mouse) of FA. The anti-FADE adduct was the major DNA adduct and was present at levels ranging from 3.5 to 37 adducts per 10(8) nucleotides (106-1138 fmols/mg DNA) in all DNA samples from the organs of FA-treated mice. The lung contained anti-FADE adduct levels approximately 2, 2.5 and 5 times higher than those in kidney, liver, and spleen plus thymus, respectively. There was no significant difference between anti-FADE adduct levels in liver and kidney; however, adduct levels were significantly higher than those in spleen plus thymus. Significant variation was found among litters in anti-FADE adduct levels from lung, liver, and kidney, but not from spleen plus thymus. An unidentified peak was present in the adduct profile of the kidneys of treated but not control animals. Three additional unidentified radioactive peaks were present in adduct profiles of DNA from tissues of both treated and control animals, but their levels were significantly higher in the tissues of treated animals than in controls. Possible correlations between tumorigenic response and DNA adduct formation are discussed.

Animals↗
PubMed1995

Formation and persistence of DNA adducts in organs of CD-1 mice treated with a tumorigenic dose of fluoranthene.

Fluoranthene (FA) is tumorigenic to the lung when injected i.p. into CD-1 mice 1, 8 and 15 days after birth (Wang, J.-S. and Busby, W.F. Jr, Carcinogenesis, 14, 1871-1874, 1993). Levels, tissue distribution and persistence of FA--DNA adducts detected by HPLC-32P-postlabeling were investigated during the course of lung tumorigenesis by FA. Anti-10b-N2-deoxyguanosin-1,2,3,-trihydroxy-1,2,3 10b-tetrahydrofluoranthene [sequence: see text] (anti-FADE adduct) was consistently the major adduct in DNA samples from lung, heart, liver and kidney of animals examined at different time points from 2 h to 165 days after the last treatment with the tumorigenic dose (3.5 mg/mouse) of FA. Several unidentified adducts were also detected. Lung, the target organ for FA tumorigenicity, contained higher levels of anti-FADE adduct than other tissues from 1-165 days after treatment. The anti-FADE adduct level decreased in a biphasic manner after reaching maximum values at 2 h in heart and spleen plus thymus and 3 days in lungs, liver and kidneys. About 10% of the maximum amount of anti-FADE adduct remained in lung, liver and heart 165 days after final FA treatment, at which time 44% of animals had developed lung adenomas. Significant inter-litter variations, but no sex differences in adduct levels were observed. These results indicated a positive correlation between anti-FADE adduct level and persistence in relation to target organ specificity for tumor formation.

Animals↗
PubMed1994

Molecular biomarkers for aflatoxins and their application to human cancer prevention.

The rapidly expanding understanding of the progressive processes of carcinogenesis provides opportunities for the identification of molecular biological markers reflecting events from exposure through clinical disease. These molecular biological markers can be classified into categories of markers of exposure reflecting the dose of toxic agents, markers of effect indicating a biological response to exposure, and markers of susceptibility providing information about the inherent sensitivity of individuals to the toxic agents. By definition some of these markers are chemical agent specific, such as a carcinogen-DNA or -protein adduct, while others are biological process specific, such as the altered expression of a gene. This article reviews the development and validation of molecular biomarkers of aflatoxins using experimental and human population studies. The development of molecular biomarkers for aflatoxins is based upon the extensive research database available about their metabolism, macromolecular adduct formation, and general mechanisms of action. The long-term goal of the research described in this paper is the application of aflatoxin biomarkers to the development of preventive interventions in human populations at high risk for liver cancer.

Aflatoxins↗
PubMed1994

Adduct detection by acylation with [35S]methionine: analysis of DNA adducts of 4-aminobiphenyl.

Reaction of synthetic N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (dGuo-8-ABP) with t-butoxycarbonyl-L-[35S]methionine, N-hydroxysuccinimidyl ester (35S-labeled TBM-NHS), under optimized conditions produced mono-, bis-, and tris-TBM-acylated nucleosides that were separable by HPLC. Reaction of different amounts of N-(2'-deoxy-1',2'-[3H]guanosin-8-yl)-4-aminobiphenyl ([3H]dGuo-8-ABP) with 35S-labeled TBM-NHS established that total 35S content of acylated products was linearly related to adduct concentration (r = 0.992) over the range of 10 fmol to 30.6 pmol. Additionally, the N-(deoxyguanosin-8-yl)-4-[3H]aminobiphenyl (dGuo-8-[3H]ABP) adduct was isolated from calf thymus DNA adducted in vitro and from rat liver DNA adducted in vivo and similarly reacted with 35S-labeled TBM-NHS. Acylation products of dGuo-8-ABP from all three sources showed HPLC retention times identical to those of authentic TBM-dGuo-8-ABP, and 35S incorporation into acylated products was linearly related to amount of adduct reacted. These results indicate that the procedure, to which we have referred as adduct detection by acylation with methionine (ADAM), has potential applicability as an analytical procedure for detection and quantification of DNA adducts in human tissues in the molecular epidemiology of cancer.

Acylation↗
PubMed1993

Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli.

The repair by UvrABC endonuclease of two major adducts formed by aflatoxin B1 in DNA was found to be similar. Aflatoxin epoxide was used to generate the aflatoxin B1.N7-guanine adduct which can convert to aflatoxin B1-formamidopyrimidine adduct. The reaction of the aflatoxin B1 epoxide with DNA follows pseudo-first order kinetics. The DNA sequence-specific relative reactivity of the epoxide is the same as previously observed for aflatoxin B1 activated by liver microsomes, therefore strongly reinforcing the notion that aflatoxin B1 reacts with DNA through the epoxide intermediate. For the majority of lesion sites, a high affinity protein-DNA complex was formed from the UvrA and the UvrB proteins with similar efficiency to both adducts, and to pyrimidine dimers, and then nicks the DNA when UvrC was added. The two incisions are at the eighth phosphodiester moiety 5' and the sixth phosphodiester moiety 3' of a modified guanine nucleotide. Both incisions appeared to be concerted. For some sites, the DNA sequence can alter the relative incision efficiency up to 15-fold. However, the majority of these AFB1 lesion structures in most DNA sequences are similar with respect to recognition by this nucleotide excision repair enzyme. Therefore the observation that the aflatoxin B1.N7-guanine lesion is removed rapidly, while the aflatoxin B1-formamidopyrimidine lesion persists in the mammalian cell may have other mechanistic explanations.

Aflatoxin B1↗
PubMed1993

Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.

Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFB1)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with allele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G.C-to-A.T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G.C-to-T.A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G.C-to-T.A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepato-cellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.

Adenoma↗
PubMed1993

Modulation of c-myc gene expression in rat livers by aflatoxin B1 exposure and age.

A single dose of aflatoxin B1 (AFB1) caused a rapid and transient induction in c-myc mRNA levels in livers derived from adult male Fischer 344 rats. The inducibility of c-myc expression by AFB1 increased with age as c-myc mRNA levels from untreated livers declined from 18-fold in 29-day-old animals to 1-fold in 39-day-old animals, relative to untreated 43-day-old control animals. A dose-dependent increase in c-myc mRNA was found when 53- and 76-day-old rats were administered various AFB1 doses whereas 29-day-old animals exhibited essentially no change in c-myc mRNA levels for the doses examined. Rats were treated with multiple doses of AFB1 to induce hepatocellular carcinomas. C-myc mRNA levels were measured in rat liver samples taken during and subsequent to chronic AFB1 exposure. Characterization of the c-myc induction response at the time of AFB1 exposure revealed that a transient elevation of c-myc mRNA occurred during each (5-day) dosing period for the first 3 weeks, consistent with the acute exposure studies. RNA prepared from hepatocellular carcinomas exhibited elevated levels of c-myc mRNA compared to vehicle-treated control animals. Examination of tumors isolated from the 28-day-old animals showed that the increased c-myc mRNA observed was not the result of gene amplification or gene rearrangement. A comparison of tumor data showed that the 28-day-old animals had a 100% liver tumor incidence while the 38-day-old rats had a 20% liver tumor incidence.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMed1993

Quantitative analysis of aflatoxin-albumin adducts.

Quantitative exposure assessments made using biologically relevant markers will facilitate epidemiological studies of risk from environmental carcinogens. Blood proteins are readily accessible macromolecules that have been shown to be targets for activated chemical carcinogens. Serum albumin is quantitatively the most abundant target for aflatoxin B1 and the measurement of aflatoxin-serum albumin adducts has been used to detect exposed individuals. The goal of these experiments was to devise an analytical procedure that would increase the overall recovery of aflatoxin adducts in serum albumin, and thereby improve the accuracy of exposure monitoring. The method developed consisted of the following procedures. Proteins were precipitated from serum (< or = 100 microliters) with 80% ammonium sulfate, with incubation at 4 degrees C for 2 h. Following dialysis against phosphate-buffered saline (pH 7.0 for 3 h at 4 degrees C), the proteins were digested with protease (Pronase) (1:4.1 w/w enzyme:protein) for 15 h at 37 degrees C with shaking. Enzyme and other undigested proteins were precipitated with acetone (1:2 v/v, 40 min, 4 degrees C). After evaporation of the acetone under vacuum, levels of aflatoxin B1-albumin adducts were determined by radioimmunoassay carried out on 300 microliters fractions. This procedure obviated the isolation of albumin prior to analysis and reduced interference in the radioimmunoassay. High recoveries of aflatoxin B1 adducts were achieved together with a low limit of detection. The applicability of the procedure in epidemiological studies of human aflatoxin exposure was illustrated by results of analysis of aflatoxin-albumin adducts in serum samples from residents of Chongming Island, People's Republic of China.

Aflatoxin B1↗
PubMed1993

Molecular epidemiology of aflatoxin exposures: validation of aflatoxin-N7-guanine levels in urine as a biomarker in experimental rat models and humans.

Human epidemiology and experimental animal data have provided the statistical association and biological information necessary to propose that aflatoxins are risk factors for human liver cancer. As liver cancer causes at least 200,000 deaths per year, prevention measures must be developed to ameliorate this nearly always fatal disease. Preventive strategies will be facilitated by the identification of individuals at high risk. It is the goal of the molecular dosimetry field to provide facile and accurate biomarkers to identify people at high risk for carcinogen exposure and consequent adverse health effects. We have developed methods to defect the major aflatoxin DNA adduct, aflatoxin N7-guanine (AFB-N7-guanine), in urine, examined the dose-response characteristics in people living in China and The Gambia, and have found an excellent association of this biomarker with exposure. In addition to exposure studies in people, our laboratories have monitored AFB-N7-guanine excretion in the urine of rats whose risk for developing cancer has been modulated with dietary chemoprotective agents such that independent groups of animals receiving the same dosage of aflatoxin B1 were at either high or low risk for tumorigenesis. The production of DNA damage by aflatoxins is not the exclusive mechanism for liver cancer. Many other factors, including hepatitis B virus, cell proliferation, and nutritional status, can exert strong modification effects in human disease. Thus, molecular epidemiological investigations that examine only one biomarker may greatly underestimate or overestimate the risk for an individual.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma.

Aflatoxins have long been suspected to be human hepatic carcinogens but no direct study was feasible until assays to measure individual aflatoxin exposure became available. We have used assays for urinary aflatoxin B1, its metabolites AFP1 and AFM1, and DNA-adducts (AFB1-N7-Gua) to assess the relation between aflatoxin exposure and liver cancer, as part of an ongoing prospective study of 18,244 middle-aged men in Shanghai, People's Republic of China. After 35,299 person-years of follow-up, 22 cases of liver cancer had been identified. For each case, 5 or 10 controls were randomly selected from cohort members without liver cancer on the date the disorder was diagnosed in the case and matched to within 1 year for age, within 1 month for sample collection, and for neighbourhood of residence. Subjects with liver cancer were more likely than were controls to have detectable concentrations of any of the aflatoxin metabolites (relative risk 2.4, 95% confidence interval 1.0-5.9). The highest relative risk was for aflatoxin P1 (6.2, 1.8-21.5). In an analysis adjusting for the effects of hepatitis B surface antigen seropositivity, level of education, cigarette smoking, and alcohol consumption, the relative risk for the presence of aflatoxin metabolites was 3.8 (1.2-12.2). There was a strong interaction between serological markers of chronic hepatitis B infection and aflatoxin exposure in liver-cancer risk. Reduction of aflatoxin exposure may be a useful intermediate goal in prevention of liver cancer, since the benefits of wide-scale hepatitis B vaccination will not be apparent for many years.

Aflatoxins↗