Leishmania donovani: in vitro growth inhibition of promastigotes by a carbocyclic analog of 2'-deoxyguanosine.
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Animals↗
Biomedical subjects
A M Jeffrey
Publications and source records attributed to A M Jeffrey.
Stability of fusarin C: effects of the normal cooking procedure used in China and pH.
Fusarin C (FC) is a naturally occurring mutagen found on Fusarium moniliforme-contaminated corn. In Linxian, China, an area of exceptionally high incidence of esophageal cancer, the milled corn is made into small cakes called wotou, which are steamed. It has been suggested that FC may play a role in the etiology of esophageal cancer, but this has been questioned, in part, because of the thermal instability of FC. We therefore measured the effects of temperature on FC stability, mimicking the cooking process. FC was extracted from the wotou and quantitated by both reverse-phase high-performance liquid chromatography and the Ames assay. After wotou were steamed for 30 minutes, 11% and 3% of the initial FC remained, as detected by the two assays, respectively. The milled corn, when moistened with water, had a pH of 6.1, which was lowered to 5.3 when it was inoculated with F. moniliforme and cultured for three weeks. Pickled vegetables are sometimes added to the wotou, which further reduces the pH. To determine the effects of pH on FC, it was heated in solution, with the pH varied between 4 and 8. Its stability was very pH dependent, decomposing more rapidly as the pH increased. Because ingested FC would also be subjected to the low pH of the stomach, it was treated for 30 minutes at 37 degrees C and pH values ranging from 1.0 to 7.0. FC was stable (less than 15% decomposition) under these conditions. If FC is a significant health threat, it could most easily be destroyed by cooking at slightly basic pH values.
China↗
Biologic markers in ethylene oxide-exposed workers and controls.
Ethylene oxide (EtO) is an alkylating agent and a model direct-acting mutagen and carcinogen. This study has evaluated a panel of biologic markers including EtO-hemoglobin adducts (EtO-Hb), sister-chromatid exchanges (SCEs), micronuclei, chromosomal aberrations (CAs), DNA single-strand breaks (SSB) and an index of DNA repair (ratio of UDS to NA-AAF-DNA binding) in the peripheral blood cells of 34 workers at a sterilization unit of a large university hospital and 23 controls working in the university library. Comprehensive environmental histories were obtained on each subject including detailed occupational and smoking histories. Industrial hygiene data obtained prior to the study and personal monitoring during the 8 years preceding the study showed that workers were subject to low-level exposure near or below the current Occupational Safety and Health Administration (OSHA) standard of 1 ppm (TWA). Personal monitoring data obtained during 2 weeks prior to blood sampling were uniformly less than 0.3 ppm (TWA). After adjusting for smoking, EtO workplace exposure was significantly (p less than 0.001) associated with EtO-Hb (a carcinogen-protein adduct) and 2 measures of SCEs [the average number of SCEs/cell (SCE50) and the number of high frequency cells (SCEHFC)]. There was an apparent suppression of DNA repair capacity in EtO-exposed individuals as measured by the DNA repair index; i.e., the ratio of unscheduled DNA synthesis (UDS) and NA-AAF-DNA binding (p less than 0.01). No association of DNA repair index with smoking was found. Another important finding of this study is the highly significant correlation between EtO-Hb adduct levels and SCEHFC (p less than 0.01) and SCEs (p less than 0.02) which provides evidence of a direct link between a marker of biologically effective dose and markers of genotoxic response. In contrast, micronuclei, CAs and SSBs were not significantly elevated in the workers. The activity of the u-isoenzyme of glutathione-S-transferase (GT) was measured as a possible genetic marker of susceptibility and a modulator of biomarker formation. However, possibly because of confounding by age, no significant relationships were found between GT and any of the exposure-related markers by ANOVA or among other independent variables by regression. This study demonstrates significant effects of low-level EtO exposure, independent of smoking history, near or below 1 ppm on multiple biomarkers and suggests that the current OSHA standard may not be adequately protective. Previously described effects of smoking on EtO-Hb adducts, SCEs and SCEHFC were also seen in this study.(ABSTRACT TRUNCATED AT 400 WORDS)
Adult↗
Biomarkers in styrene-exposed boatbuilders.
14 fiberglass-reinforced plastics (FRP) boatbuilders were compared with 9 unexposed controls with respect to several chemical specific and nonspecific biomarkers measured in peripheral blood. Biomarkers included styrene-hemoglobin adducts (styrene-Hb), sister-chromatid exchanges (SCEs), micronuclei (MN), single-strand breaks (SSBs) and N-acetoxy-2-acetylaminofluorene-induced DNA binding (NA-AAF binding) as a measure of susceptibility to DNA damage. Workers' exposures averaged 11 ppm (8-h TWA; geometric mean) and ranged from 0.6 to 44 p.p.m. Mandelic acid levels were measured in end-of-shift urine samples and reflected an average styrene exposure equivalent to 15 p.p.m. There was a large though not significant difference in levels of styrene-Hb adducts among exposed workers and controls, largely the consequence of a single heavily-exposed individual with an extremely high level of adducts. Significant differences between biomarker levels in exposed workers and controls were observed with MN, SSBs and NA-AAF binding. No significant differences were seen in mean levels of SCEs nor in the incidence of cells with a high frequency of SCEs. The data suggest that exposure to levels of styrene in occupational settings near or below the current OSHA standard (50 p.p.m.) can induce damage at the cellular/molecular level. Appropriately-selected panels of biomarkers can be useful in identifying potentially harmful exposures.
Acetoxyacetylaminofluorene↗
Hydrolysis of carcinogen--DNA adducts by three classes of deoxyribonucleosidase to their corresponding bases.
We have investigated three different enzymes, each of which splits the glycosidic bonds of normal deoxyribonucleosides, for their ability to cleave acid-sensitive carcinogen-DNA adducts: Leishmania donovani purine deoxyribonucleosidase; Lactobacillus helveticus trans-N-deoxyribosylase; and purine nucleoside phosphorylase. Twenty-eight substrates were assayed using one or more of the enzymes, monitoring free base formation by reverse-phase HPLC and a diode array detector. Products were characterized by their retention times and UV spectra. The deoxyribonucleosidase showed the broadest specificity: substrates cleaved by either the trans-N-deoxyribosylase or the phosphorylase were hydrolyzed by the deoxyribonucleosidase. O6- and N2-alkyldeoxyguanosine, N6-alkyl deoxyadenosine and several cyclic adducts were all hydrolyzed by the deoxyribonucleosidase. None of these enzymes cleaved three bulkier adducts; N2-, N6- and C-8 derivatives of benzo[a]pyrene, benzo[c]phenanthrene and N-acetoxy-2-acetylaminofluorene, respectively. Only the deoxyribonucleosidase hydrolyzed O6-methyldeoxyguanosine while both the deoxyribonucleosidase and trans-N-deoxyribosylase cleaved N6-methyl deoxyadenosine. These enzymes, especially the deoxyribonucleosidase, provide mild ways to release the carcinogen-modified base from deoxyribose and will be useful in DNA adducts analysis.
Alkylation↗
Mechanisms of organ specificity in chemical carcinogenesis.
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Carcinogens↗
Metabolic activation of nitropyrenes and diesel particulate extracts.
The aim of this research was to investigate the possible risks of genotoxicity associated with human exposure to diesel engine emissions. We sought to identify and evaluate the critical components of such emissions by using a variety of short-term biological systems. Adducts formed between benzo[a]pyrene and DNA in several short-term test systems have been thoroughly investigated. Although benzo[a]pyrene has long been used as an index of the potential carcinogenicity of polycyclic aromatic hydrocarbon mixtures and is present in diesel engine emissions, it may not be the best measure of the carcinogenicity of these emissions if, indeed, they are confirmed to exert such an effect in humans. Certain nitroarenes, known to be present in diesel particulate extracts, are very potent mutagens in the Ames assay. The major adducts formed in Salmonella typhimurium with 1-nitropyrene (Howard and Beland 1982) and 1,8-dinitropyrene (Andrews et al. 1986; Fifer et al. 1986) have been identified. We undertook, therefore, a comparison of the DNA adducts formed between 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene and cellular DNA in various systems, including human bronchial segments, in rabbit lung and trachea, and mouse embryo fibroblast C3H/10T1/2 cells, with those reported to be formed in S. typhimurium. In these studies, we administered radiolabeled nitropyrenes, analogous to the treatments previously employed for testing benzo[a]pyrene, and isolated and digested the modified DNA. We then compared elution times, by high-pressure liquid chromatography, of the radioactive adducts with synthetic standards. Not all combinations of exposures were undertaken, since the direction of the investigations changed to include studies on adducts formed in animals exposed to diesel engine emissions themselves. Of the samples studied, metabolism of 1-nitropyrene was most evident in the human bronchial tissue. Little metabolism was evident in duodenal and colonic samples. Mouse C3H/10T1/2 embryo fibroblasts showed no detectable metabolism of, DNA adduct formation with, or transformation by 1-nitropyrene. DNA adducts were detected in cells exposed to 1,8-dinitropyrene and traces of adducts were formed with either 1,3-dinitropyrene or 1,6-dinitropyrene. Measurements of DNA adducts formed after treatment with [4,5,9,10-3H]-1-nitropyrene in rabbit tracheal samples showed that only about 2 to 15 percent of the associated radiolabel could be accounted for as the previously identified C-8 adduct. To facilitate the quantification of adduct levels that might occur in humans and laboratory animals exposed to the nitroarenes in diesel engine emissions, antisera were prepared against DNA modified with 1-nitrosopyrene.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Molecular epidemiology and cancer prevention.
A review of recent studies of biologic markers in populations with model exposures to carcinogens (cigarette smoke and polycyclic aromatic hydrocarbons) illustrates their potential role in cancer prevention. Data on macromolecular adducts and oncogene activation from cross-sectional, serial sampling and case-control studies demonstrate the usefulness of biologic markers in signalling a potential carcinogenic risk and in estimating the magnitude of interindividual variation within exposed groups. Recommendations for future research include nested case-control studies to establish the relationship between markers of biologic dose and effect (e.g., adducts, gene mutation, oncogene activation) and cancer risk.
Air Pollutants↗
Metabolism of fusarin C by rat liver microsomes. Role of esterase and cytochrome P-450 enzymes with respect to the mutagenicity of fusarin C in Salmonella typhimurium.
Fusarin C (FC) is a potent mutagen present on Fusarium moniliforme contaminated corn. This compound requires metabolic activation for which microsomes from phenobarbital-induced rats are most effective. Inhibition of the simultaneously induced esterase activity, which produced a less mutagenic metabolite, doubled the mutagenicity of FC. Carbon monoxide inhibited the mutagenicity of FC, suggesting the involvement of a heme containing enzyme. However, monoclonal antibodies specific for the phenobarbital-induced cytochrome P-450 enzymes PB-4 and PB-5, while inhibiting O-demethylation of p-nitroanisole and aflatoxin B1 mutagenicity, had not effect on FC mutagenicity. This implies that either these enzymes are not involved in the activation of FC or FC competes well with the antibodies for binding to the cytochrome P-450 enzymes. Two additional metabolites of FC were detected. One had an ultraviolet spectrum similar to FC: the other had a lambda max at 326 nm, and its retention time on reverse phase HPLC was very sensitive to changes in pH.
Aflatoxin B1↗
Chromatographic and spectrophotometric characterization of adducts formed during the reaction of trans,trans-muconaldehyde with 14C-deoxyguanosine 5'-phosphate.
Mice liver microsomes oxidatively open the benzene ring to form trans,trans-muconaldehyde, a hematotoxic unsaturated aldehyde. In the present studies, 4.5 mumole trans,trans-muconaldehyde was reacted with 14C-2'deoxyguanosine 5'-phosphate in phosphate buffer. Products were separated by high performance liquid chromatography (HPLC). Absorbance was monitored using a diode array detector, and aliquots of the HPLC eluant were collected for UV spectrophotometric analysis and scintillation counting. Under these conditions, deoxyguanosine 5'-phosphate eluted at 12.5 min and muconaldehyde at 22.0 min. The HPLC and radioactivity profiles of the muconaldehyde/deoxyguanosine reaction mixture indicated the presence of multiple adducts. Three adducts were detected eluting at 36, 39, and 42 min, which represented approximately 2.5, 2.5, and 1% of the radioactivity, respectively. These adducts had similar UV spectra with absorption maxima between 334 and 347 nm. Another product of the reaction mixture, eluting at 19.0 min and accounting for 10% of the radioactivity, was also observed. This compound had absorption maxima at 348 and 372 nm. These results suggest that trans,trans-muconaldehyde can react with deoxyguanosine monophosphate in vitro under physiological conditions to form stable adducts. Studies are being conducted to determine the structure of these adducts and whether these adducts are formed by the reaction of DNA with muconaldehyde or metabolically activated benzene.
Aldehydes↗
Fusarium moniliforme metabolites: genotoxicity of culture extracts.
Fusarin C (FC) is a potent mutagen which has been isolated from Fusarium moniliforme culture extracts (FME). We have confirmed that the mutagenicity of these extracts is enhanced by phenobarbital- or Aroclor-induced microsomes and shown that: (i) additional, direct-acting, mutagens are present in crude extracts from F. moniliforme cultures; (ii) Salmonella typhimurium TA 100 exposed to FME in the presence of S9 mixtures shows an increased number of DNA strand breaks as detected by intercalation of ethidium bromide; (iii) exposure of polyoma-transformed rat fibroblast cells to HPLC-purified FC induced asynchronous replication of polyoma DNA sequences, a phenomenon also observed when these cells were exposed to a variety of other carcinogens; (iv) FME can alkylate 4-(p-nitrobenzyl)pyridine in the absence of S9 mix, although less efficiently than styrene oxide; and (v) these additional direct-acting mutagens, present in crude extracts from F. moniliforme cultures, may be responsible for the DNA adducts formed by reaction with calf thymus DNA in the absence of metabolic activation and detected by the 32P-postlabeling assay. All of these observations suggest that significant health effects may be associated with human exposure to F. moniliforme and that further studies on its metabolites are needed.
Animals↗
Enhancement of sensitivity of fluorescence line narrowing spectrometry for detection of carcinogen-DNA adducts.
Fluorescence line narrowing (FLN), is a method by which highly characteristic spectra have been obtained for a large number of polycyclic aromatic hydrocarbon (PAH)-DNA adducts and is well suited for the analysis of exposures to complex mixtures of PHA. The basic method is described and recent improvements discussed which overcome one of the major limitations of the method to its wider application to biological samples, that is its sensitivity.
Animals↗
Fatty acid modification of C3H 10T 1/2 fibroblast cells: changes in benzo(a)pyrene metabolism and phorbol ester binding.
The mouse embryo fibroblast cell line, C3H 10T 1/2 Cl8, was studied as an in vitro experimental model to investigate the mechanism and specificity behind the modulation of carcinogenesis by dietary lipid. The cells were grown in medium supplemented with 95 microM stearate, linoleate, or palmitate as fatty acid/albumin complexes, during which time they maintained normal growth and morphology characteristics. After 5 days of supplementation total cellular lipid fatty acid was enriched in the supplemented fatty acid. By Day 40, however, fatty acid profiles of all groups were the same. Cellular uptake and utilization of 14C-radiolabeled fatty acids were measured. Within 24 h of supplementation, label was incorporated into cholesterol and diglycerides, cholesterol ester, alkyldiacylglycerols, and phospholipids. Approximately half of the radiolabel was found in phosphatidylcholine. Supplementation significantly increased the rate of benzo(a)pyrene metabolism, but did not affect DNA modification by benzo(a)pyrene. Phorbol dibutyrate binding to C3H 10T 1/2 cells at 4 degrees C was modified by lipid supplementation. At 37 degrees C and 23 degrees C, phorbol dibutyrate binding was characterized by both high- and low-affinity sites for linoleate- and stearate-supplemented cells. At 4 degrees C high-affinity binding was absent in stearate- and palmitate-supplemented cells, but was maintained in linoleate-supplemented cells. These studies suggest that the unsaturated fatty acid content of the diet may not significantly affect the initiation stage of benzo(a)pyrene carcinogenesis, but may instead affect promotion. One possible mechanism for this could involve changes in the lipid microenvironment of membrane receptors involved in tumor promotion.
Animals↗
Investigation of benzo[a]pyrene-globin adducts.
The nature of the adducts formed between benzo[a]pyrene (BP) and globin were investigated in animals treated with [3H]BP. Modification levels on globin were determined by radioactivity measurements. Since BP tetraols can be released from benzo[a]pyrene diol epoxide modified protein and DNA by acid treatment, globin samples were treated with acid, released tetraols separated by HPLC and quantitated by scintillation counting. In addition, acid released material was measured in a competitive enzyme linked immunosorbent assay (ELISA) using antibodies which recognize BP tetraols. Both measurements indicate that only 2% of bound radioactivity could be released as free BP tetraols. These studies indicate that benzo[a]pyrene diol epoxide may not be the major metabolite of BP involved in globin binding.
Animals↗
Covalent binding of ethylated analogs of 7,12-dimethylbenz[a]anthracene to the DNA of mouse embryo fibroblast 10T1/2 cells.
The carcinogenicity of a number of ethylated analogs of 7,12-dimethyl-benz[a]anthracene (DMBA) has been previously studied. The covalent binding of these compounds to mouse embryo fibroblast 10T1/2 cell DNA expressed as adducts/10(6) bases, paralleled the original carcinogenicity studies and in decreasing order of potency were DMBA, 7-ethyl-12-methylbenz[a]anthracene (7-Et,12-MeBA), 7-methyl-12-ethylbenz[a]anthracene (7-Me,12-Et), and 7,12-diethylbenz[a]anthracene (7,12-DEBA). The results demonstrate that a major determinant of the carcinogenicity of these compounds relates to their ability to bind to DNA.
9,10-Dimethyl-1,2-benzanthracene↗
Identification of polycyclic aromatic hydrocarbon metabolites and DNA adducts in mixtures using fluorescence line narrowing spectrometry.
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DNA↗
Computer-assisted molecular modeling of tumor promoters: rationale for the activity of phorbol esters, teleocidin B, and aplysiatoxin.
In the two-stage model of skin carcinogenesis, it is believed that initiators bind to DNA and that tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA) bind noncovalently to membrane-associated high-affinity receptors, probably protein kinase C. Two other types of potent tumor-promoting substances, aplysiatoxin and teleocidin, appear to act also by binding to and activating protein kinase C, even though their chemical structures are quite different. Therefore, we have undertaken computer modeling of the special relationship of various functional groups in these three chemical classes of tumor promoters in an attempt to explain how these diverse structures bind to the same receptor molecule. We propose a stereochemical model in which the oxygens in TPA at C-3, C-4, C-9, and C-20 (O-3, O-4, O-9, and O-20) correspond to the O-11, N-13, N-1, and O-24 positions in teleocidin and the O-27, O-3, O-11, and O-30 oxygens in aplysiatoxin, respectively. In this model all distances with respect to overlap of the corresponding atoms are less than 1 A. In addition, all three types of molecules have their hydrophobic moieties oriented in a similar position. This model is further discussed with respect to other compounds showing various degrees of activity as tumor promoters, including mezerein, ingenol, and 4 alpha-TPA. The model explains how chemically diverse structures can have similar biological activity as tumor promoters and provides a basis for designing both agonists and antagonists of tumor promoters.
Carcinogens↗
Identification of DNA damage as a result of exposure of rats to diesel engine exhaust.
Owing to the concern regarding genotoxic agents which may be present in diesel engine emissions we have studied the effect, with respect to DNA damage, of the exposure of F344 rats to such emissions for approximately 31 months at levels of particulate material of 7.1 mg/m3. Rats were killed and the DNA extracted from the right lung lobe and coded prior to analysis for DNA adducts by the 32P-postlabeling technique. Although a gradation of adduct intensity was found from high levels to none being detectable, distinction could be made between exposed and unexposed groups based upon the intensity of DNA adducts present.
Animals↗