Biomedical subjects
S De Flora
Publications and source records attributed to S De Flora.
Assessment of antimutagenicity and anticarcinogenicity.
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Modulation of the mutagenic response in prokaryotes.
Short-term tests investigating genetic end-points in prokaryotes have been extensively used worldwide not only for risk assessment purposes but also for evaluating the modulation of the mutagenic response. In spite of some intrinsic limitations, such as the lack of cell compartmentalization or the need for an exogenous metabolic system working extracellularly, experimental systems in bacteria can provide useful preliminary indications and some information on the mechanisms involved. In the large majority of studies the putative modulator is mixed with a known mutagen and then assayed in target bacteria, with suitable controls. However, under natural conditions exposure of target cells to modulators may either precede, co-exist with, or follow exposure to mutagens. Therefore, a variety of methodological variations, involving pre-treatment, co-treatment, or post-treatment of bacteria with the putative modulator, have been designed. Application of these procedures showed that the effects of modulators can be completely upset, from inhibition to enhancement, or vice versa, by changing the experimental conditions. Use of methodological variations may provide more complete information on the spectrum of possible effects in bacteria as well as a better insight into modulation mechanisms. Several examples illustrating the flexibility of the Salmonella test in this field of research are available. On the other hand, the widespread use of these relatively simple techniques, yet requiring skillfulness and experience, may lead to some misuse or oversimplifications. A rather common inadequacy is to use excessive amounts of test mutagens, or to express the results in terms of revertants/survivors, rather than revertants/plate. In fact, in the Salmonella test the number of revertants is rather unrelated to the initial number of plated bacteria, provided a normal background lawn of bacterial growth is formed. Thus, a 50% killing of bacteria will not appreciably influence the number of revertants/plate, but expressed as revertants/survivors the effect will look twice as large.
Chemoprevention of smoke-related DNA adduct formation in rat lung and heart.
The formation of smoke-related DNA adducts and their chemoprevention were investigated in tissues of male Sprague-Dawley rats, by testing a total of 132 DNA samples by synchronous fluorescence spectrophotometry (SFS), which mainly detects benzo[a]pyrene diolepoxide (BPDE)-DNA adducts. Groups of six animals each were exposed whole-body to mainstream cigarette smoke, once daily, for up to 40 consecutive days. No adduct was revealed in liver DNA, whereas smoke-related DNA adducts were detectable in the lung from the 8th day of exposure and continued to increase until the 40th day. Adducts to heart DNA, which were monitored after 28 and 40 days of exposure, attained even higher levels than those detected in the lungs of the same animals. A high correlation existed between the amounts of smoke-related DNA adducts measured in these two organs. The daily administration by gavage of the thiol N-acetyl-L-cysteine (NAC), an effective mutation and cancer chemopreventive agent, which had been previously shown to inhibit the formation of SFS-positive DNA adducts in rats receiving intratracheal instillations of benzo[a]pyrene, significantly prevented occurrence of the same adducts in both heart and lungs of smoke-exposed rats. No fluorescence signal was observed in liver, lung, or heart DNA of sham-exposed animals. The findings of this molecular dosimetry study complement the results of parallel histopathologic, cytogenetic, biochemical and metabolic analyses of tissues and cells from the same rats, providing evidence for a variety of significant alterations produced by exposure to cigarette smoke and for the specific protective role of NAC.
Reduction of chromium(VI) to chromium(V) by rat liver cytosolic and microsomal fractions: is DT-diaphorase involved?
Incubation of rat liver cytosolic or microsomal fractions with chromium(VI) led to a dramatic decrease in chromium(VI) mutagenicity, as determined by the Ames Salmonella assay using the TA100 tester strain. The cytosol-dependent decrease in chromium(VI) mutagenicity was found to be counteracted in the presence of dicumarol, an inhibitor of the cytosolic enzyme NAD(P)H:quinone oxidoreductase (DT-diaphorase). In order to determine whether DT-diaphorase is a significant factor in enzymatic reduction of chromium(VI) in rat liver tissue, cytosolic and microsomal fractions were analyzed for NAD(P)H-dependent chromium (VI) reductase activity leading to chromium(V) formation by using electron paramagnetic resonance (EPR) spectroscopy. Reaction of chromium(VI) with NADH or NADPH in the presence of either cytosolic or microsomal fractions led to the formation of stable chromium(V)--NAD(P)H complexes. When glucose 6-phosphate (G6P) was present in the reaction as part of a NADPH-generating system, stable chromium(V)--G6P complexes were formed in addition to the chromium(V)--NAD(P)H complexes. The chromium(V) complexes had g values of 1.980-1.982 and superhyperfine splitting constants of 0.8-0.9 characteristic of bis(diol)oxochromium(V) complexes. Inhibition of 90% of the cytosolic DT-diaphorase activity by dicumarol led to only partial (20-22%) inhibition of chromium(V) formation. Visible and EPR spectroscopic studies showed that purified DT-diaphorase had no detectable chromium(VI) reductase activity and did not catalyze formation of chromium(V). Inhibition of 69% of microsomal aryl hydrocarbon hydroxylase activity by ketoconazole led to partial (10%) inhibition of chromium(V) formation. These results indicate that intracellular NAD(P)H-dependent enzymatic reduction of chromium(VI) in rat liver cannot be attributed to the activity of any one enzyme in the cytosolic or microsomal fractions. DT-diaphorase appears to play an indirect role in decreasing chromium(VI)-induced mutagenicity in Salmonella, possibly through interaction with other redox active cellular components. The involvement of diols such as sugars and pyridine nucleotides in stabilizing intracellularly generated chromium(V) is discussed.
Metabolic alterations produced by cigarette smoke in rat lung and liver, and their modulation by oral N-acetylcysteine.
Male Sprague-Dawley rats were exposed whole-body to the mainstream smoke produced by a commercial filter cigarette for 8 consecutive days, accounting for a cumulative exposure to the smoke of 75 cigarettes. Liver and lung S12 fractions were used in the Salmonella mutagenicity test in order to assess either the decrease of potency of a direct-acting mutagen (sodium dichromate) or the metabolic activation of promutagens, including cigarette smoke itself and its condensate, benzo[a]pyrene and its 7,8-diol, the aromatic amine 2-aminofluorene, and the heterocyclic amine 3-amino-1-methyl-5H-pyrido(4,3)indole. Moreover, individual biochemical parameters were measured in the liver and lung of the same rats and, in the case of cytochrome P-450-dependent monooxygenases, also in the heart of untreated or Aroclor-treated rats. The monitored biochemical parameters included aryl hydrocarbon (benzo[a]pyrene) hydroxylase and ethoxyresorufin deethylase in microsomal fractions, epoxide (benzo[a]pyrene-4,5-oxide) hydrolase in both microsomal and cytosolic fractions, glutathione (GSH) and GSH S-transferase in the cytosol. Exposure to cigarette smoke resulted in a number of significant metabolic changes, as compared to sham-exposed rats. The most pronounced alterations consisted in a 2.6-fold induction of aryl hydrocarbon hydroxylase in the lung and 8-fold induction of ethoxyresorufin deethylase in the liver, and in a marked stimulation of the liver metabolic activation of all promutagens. The last effect was inhibited by the oral administration of the chemopreventive agent N-acetylcysteine. On the whole, there was a poor correlation between the monitored biochemical and mutagenicity endpoints.(ABSTRACT TRUNCATED AT 250 WORDS)
Carcinogen-DNA adducts in human alveolar macrophages.
The occurrence of 7 beta,8 alpha-dihydroxy-(9 alpha,10 alpha)-epoxy-7,8,9,10- tetrahydrobenzo(a)pyrene (BPDE-)-DNA adducts was investigated by synchronous fluorescence spectrophotometry in human pulmonary alveolar macrophages (PAM) collected by bronchoalveolar lavage. In preliminary assays BPDE-DNA adducts were consistently detected in PAM of smokers, whereas samples from nonsmokers did not exhibit specific peaks.
Micronuclei in human alveolar macrophages.
Occurrence of micronuclei was monitored in pulmonary alveolar macrophages collected from 31 individuals undergoing diagnostic bronchoalveolar lavage. The overall frequency of micronucleated cells was 3.88 +/- 1.84/1000, without any significant difference attributable to sex, age, pathology, occupation, or smoking habits. The lack of influence of cigarette smoke on this clastogenicity index presumably reflects the very low rate of mitoses of macrophages in the alveolar lumen.
Carcinogen metabolism in human lung tissues and the effect of tobacco smoking: results from a case--control multicenter study on lung cancer patients.
Cigarette smoking is the strongest risk factor for lung cancer, but genetically determined variations in the activities of pulmonary enzyme that metabolize tobacco-derived carcinogens may affect individual risk. To investigate whether these enzymes (e.g., CYP1A-related) can serve as markers for carcinogen-DNA damage, lung tissue specimens were taken during surgery from middle-aged men with either lung cancer or non-neoplastic lung disease. Phase I [aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD)] and phase II (epoxide hydrolase, UDP-glucuronosyltransferase, glutathione S-transferase) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were also analyzed for DNA adducts, using 32P-postlabeling. The data were then analyzed for the following: a) differences in metabolic profiles between bronchial and parenchymal lung tissue; b) the effect of recent exposure to tobacco smoke on enzyme inducibility and benzo[a]pyrene metabolism; c) differences in enzyme inducibility between lung cancer and non-lung cancer patients; d) the effect of smoking on metabolism of mutagens in vitro; e) pulmonary DNA adduct levels and AHH activity in lung parenchyma of smokers and ex-smokers; f) lipid peroxidation products in lung tissue from lung cancer and non-lung cancer patients, as related to smoking habits and degree of airway obstruction; and g) prognostic value of AHH pulmonary activity in lung cancer patients. The results demonstrate a pronounced effect of tobacco smoke on pulmonary metabolism of xenobiotics and prooxidant state and suggest the existence of a metabolic phenotype at higher risk for tobacco-associated lung cancer.
Antioxidant activity and other mechanisms of thiols involved in chemoprevention of mutation and cancer.
Our studies provide evidence that thiols, such as N-acetyl-L-cysteine, inhibit both spontaneous mutations and induced mutations in bacteria, prevent the in vivo formation of carcinogen-DNA adducts, and suppress or delay the development of tumors or preneoplastic lesions in rodents. N-Acetylcysteine and other thiols exert antioxidant activity toward superoxide anion, hydrogen peroxide, and singlet oxygen, assessed in bacterial genotoxicity models. In addition, several other mechanisms were shown to contribute to their antimutagenic and anticarcinogenic activities, in the extracellular environment and in nontarget or target cells. These mechanisms include blocking of electrophilic metabolites and of direct-acting compounds, either of endogenous or exogenous source, modulation of several xenobiotic-metabolizing pathways, and protection of DNA-dependent nuclear enzymes. Chemoprevention of mutation and cancer by thiols is particularly useful under conditions of reduced glutathione (GSH) depletion due to toxic agents or to cancer-associated viral diseases, such as acquired immunodeficiency syndrome (AIDS) or viral hepatitis B.
Genotoxic, carcinogenic, and teratogenic hazards in the marine environment, with special reference to the Mediterranean Sea.
Genotoxic, carcinogenic, and teratogenic hazards arising out of pollution in the marine environment are discussed in this article, with special reference to the situation in the Mediterranean area. A number of chemical compounds or complex mixtures relevant to marine pollution, either natural or of anthropogenic origin, are tentatively listed, along with protective factors which may play a counteracting role in the same environment. Harmful substances tend to undergo interactions and transformations in seawater, sediments, and marine biota, due to physical, chemical, microbial, or light-mediated mechanisms. Bioaccumulation phenomena in marine organisms may result from food-chain biomagnification processes or from concentration of pollutants by filter feeders. A variety of sources can account for marine pollution by genotoxic, carcinogenic, and teratogenic compounds, but there is a relative paucity of analytical data concerning the Mediterranean. Metabolic transformations of xenobiotics occur in all marine organisms, the biochemical mechanisms in fish being comparable to those which have been extensively investigated in mammals. Induction of metabolic pathways, and especially of the mixed-function oxygenase system, represents the earliest warning signal of exposure to pollutants. Occurrence of neoplastic diseases is documented by experimental and field studies in marine vertebrates as well as in invertebrates. The association with local pollution phenomena has been recognized in several studies, but other etiopathogenetic factors may be also involved, and in some cases tumors have been reported to be unrelated to chemical pollution. Genotoxic agents have been detected by means of suitable techniques in seawater, sediments, and marine organisms. Several studies have investigated the presence of carcinogen-DNA adducts, DNA damage and repair processes, and cytogenetic alterations, such as chromosomal aberrations, sister-chromatid exchanges, and micronuclei, in tissues of marine organisms. However, monitoring of these end-points under field conditions encounters some limitations and problems. Even more fragmentary is the information on teratogenic effects in marine organisms, although interesting test systems have been set up. On the whole, a quite extensive database on all these toxicological issues is already available in the literature, but further studies are warranted for an adequate assessment of genotoxic, carcinogenic, and teratogenic hazards, and possibly counteracting factors in the marine environment, and specifically in the Mediterranean Sea.
Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater.
Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B1 to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment.
Benzo[a]pyrene diolepoxide-DNA adducts in alveolar macrophages of smokers.
The presence of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts was investigated by synchronous fluorescence spectrophotometry in cells recovered by bronchoalveolar lavage from 39 individuals. Pulmonary alveolar macrophages constituted, on average, 90% of these cells. No adduct was detected in samples from non-smokers or ex-smokers, whereas 84.6% of samples from current smokers exhibited typical fluorescence peaks. The samples from four subjects who had stopped smoking during the last 6 months were also positive. A considerable variability was observed in the intensity of signals, which in positive samples was significantly correlated with the number of cigarettes currently smoked but not with the cumulative total of cigarettes smoked during a lifetime. In any case, part of the recorded quantitative variations could not be ascribed to the daily number of cigarettes but probably depended on interindividual variability in smoking habits or in toxicokinetics and metabolism. On the whole, detection of BPDE-DNA adducts in alveolar macrophages, compared to other surrogate cell populations, appears to represent a sensitive and specific biomonitoring tool, assessing the internal dose of inhaled benzo[a]pyrene in the proximity of target cells of the respiratory tract. Exploitation of this end-point in molecular epidemiology and cancer prevention studies warrants further investigation.
Metabolic and genetic effects of marine pollution on aquatic organisms.
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Development and application of biomarkers exploitable for human exposure monitoring.
The objectives, applications and limitations of laboratory methods for assessing human exposure to carcinogens are concisely discussed. The available technologies include cytological, cytogenetic and molecular analyses, somatic cell mutation, carcinogen-DNA or carcinogen-protein adducts, metabolic markers, and chemical or biological analyses of expired air, body fluids, and excreta.
Classification of mechanisms of inhibitors of mutagenesis and carcinogenesis.
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Genotoxicity of chromium compounds. A review.
This article reviews approximately 700 results reported in the literature with 32 chromium compounds assayed in 130 short-term tests, using different targets and/or genetic end-points. The large majority of the results obtained with Cr(VI) compounds were positive, as a function of Cr(VI) solubility and bioavailability to target cells. On the other hand, Cr(III) compounds, although even more reactive than Cr(VI) with purified nucleic acids, did not induce genotoxic effects in the majority of studies using intact cells. Coupled with the findings of metabolic studies, the large data-base generated in short-term test systems provides useful information for predicting and interpreting the peculiar patterns of Cr(VI) carcinogenicity.
Potent genotoxicity of halogen lamps, compared to fluorescent light and sunlight.
The light emitted by halogen lamps induced mutations in Salmonella typhimurium and DNA damage in Escherichia coli, as shown by the hypersensitivity of DNA repair-deficient strains. The mutagenicity of halogen lamps was considerably higher than that of fluorescent light and of sunlight, even at much lower illuminance levels. Excision mechanisms and SOS functions were involved in repairing light-induced base-pair substitutions and frameshift errors in bacterial DNA. At variance with solar irradiation, which produces mutagenic effects over a wide UV spectrum, genotoxicity of halogen lamps was almost exclusively due to far-UV wavelengths transmissible through UV-R-250 and UV-R-280 interference filters. The main mutagenic component of fluorescent light (254 nm) were almost 10(4)-fold more mutagenic than near-UV wavelengths (365 nm). All light sources exhibited some residual mutagenicity even following filtration through various cloths. On the other hand, appropriate glass or plastic covers consistently prevented mutagenic effects. This emphasizes the urgent need for a compulsory shielding of halogen and fluorescent lamps in order to prevent unnecessary exposures to genotoxic and potentially carcinogenic UV radiations.