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Biomedical subjects

M Castegnaro

Publications and source records attributed to M Castegnaro.

At least 19 recordsLinked to original sources
PubMed1995

Formation of ochratoxin A metabolites and DNA-adducts in monkey kidney cells.

Monkey kidney cells (named Vero cells) were incubated with increasing doses of ochratoxin A (10-100 microM). The inhibiting concentration 50% (IC50) on protein synthesis was about 14 microM in the presence of 5% fetal calf serum and 37 microM in the presence of 10% fetal calf serum. Some metabolites of ochratoxin A, including the chlorinated dihydroisocoumarin moiety of OTA (OT alpha), 4-[S]-hydroxy-OTA and 4-[R]-hydroxy-OTA were detected by HPLC in the mixture of cell homogenate after a 24 h incubation with 10 and 25 microM of OTA. Using the 32P-postlabelling method, several DNA-adducts, similar to those formed in mouse kidney after OTA treatment, were detected in monkey kidney cells. Thus, Vero cells are suitable for genotoxic and cytotoxic studies in relation to the metabolism of nephrotoxic xenobiotics such as OTA.

Animals↗
PubMed1995

IARC activities in mycotoxin research.

The creation of the International Agency for Research on Cancer (IARC) in May 1965 occurred only two years after publication of the chemical structure of the aflatoxins, and the investigation of a possible link between exposure to these compounds and liver cancer incidence was initiated by IARC as early as 1968. Thus, mycotoxins were one of the first topics of research at IARC and the Agency's special interest in cancer in developing countries has contributed to a sustained effort in this research field. The work performed comprises a number of aspects including laboratory research into mechanisms of action and methods for destruction of mycotoxins, epidemiological studies, and through evaluation of the carcinogenic potency of these toxins in the "IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans." A particular feature has been the integration into epidemiological studies of biomarkers of mycotoxin exposure (e.g., to aflatoxin, ochratoxin A) developed in the IARC laboratories. The above research areas are described briefly below and future activities are discussed in relation to present knowledge and the possibilities of achieving primary prevention of cancer. Another field of activity in which IARC has been strongly involved is that of quality assurance with the organisation of the mycotoxin check sample programme [Friesen, 1989: J Toxicol-Toxin Reviews 8:363-373]. This activity will, however, not be discussed in this article in view of space limitations.

Aflatoxins↗
PubMed1995

Metabolic polymorphism affecting DNA binding and excretion of carcinogens in humans.

A case-control study on lung cancer patients demonstrated the pronounced effect of tobacco smoke on pulmonary carcinogen metabolism and suggested the existence of a metabolic phenotype at higher risk for tobacco-associated lung cancer. Lung cancer patients who were recent smokers showed in their lungs (i) significantly induced CYP1A1-related enzyme activity vs smoking non-lung cancer patients; (ii) increased benzo(a)pyrene (BP) tetrol formation from BP 7,8-diol by lung microsomes; and (iii) high levels of cytochrome P4501a1 by immunohistochemical staining. Levels of bulky aromatic DNA adducts (by 32P-postlabelling) and of BP-diol-epoxide (BPDE) adducts (by HPC/fluorometry) were quantified in lung parenchyma. Aryl hydrocarbon hydroxylase activity and the level of BPDE-DNA adducts (r = 0.91; p < 0.001) and to a lesser degree bulky DNA adducts were correlated. Thus pulmonary CYP1A1 expression (inducibility) controls in part polycyclic aromatic hydrocarbon-DNA adduct formation in tobacco smokers and, therefore, appears to be associated with lung cancer risk. High risk subjects for lung cancer among smokers may be identifiable through genotyping for polymorphic drug metabolizing enzymes in combination with molecular dosimetry of carcinogen-DNA adducts and mutation analysis in target (surrogate) cells. Such studies in a Finnish cohort of lung cancer patients and controls are in progress. Interim results of the effect of metabolic polymorphism on the level of PAH-DNA adducts and on the excretion of mutagens in urine are summarized.

Biotransformation↗
PubMed1994

Potassium permanganate can be used for degrading hazardous compounds.

Solutions of potassium permanganate in 3 M sulfuric acid, 1 M sodium hydroxide solution, and water can be used to degrade hazardous compounds. Excess oxidant can be removed by using sodium metabisulfite. Manganese, a carcinogen and mutagen, can be removed from the final reaction mixtures by making these mixtures strongly basic. Aqueous dilution causes the soluble potassium sulfate to dissolve while still allowing the insoluble manganese compounds to be removed by filtration and so reduces the weight of precipitate. In all cases the amount of manganese left in the filtrates was less than 2 ppm and the reaction mixtures were nonmutagenic. When ethanol was used as a test compound, degradation was much more rapid when the solvent was 3 M sulfuric acid or 1 M sodium hydroxide solution than when the solvent was water. However, the variation of the rate of reaction with pH depends on the nature of the substrate. Thus the effectiveness of the various methods may vary for other substrates. Potassium permanganate in sulfuric acid was used to degrade four polycyclic heterocyclic hydrocarbons. Destruction was greater than 99.9% and the final reaction mixtures contained no more than 0.5 ppm manganese and were not mutagenic. By modifying the work-up procedures to remove manganese from the final reaction mixture, procedures previously developed for degrading hazardous compounds can still be employed.

Chemical Phenomena↗
PubMed1993

Ochratoxin A-related DNA adducts in urinary tract tumours of Bulgarian subjects.

Ochratoxin A (OTA), a natural contaminant of mouldy food and feed, is suspected of being one of the etiological agents responsible for Balkan endemic nephropathy (BEN) and the associated urinary tract tumours. We have previously shown that ochratoxin A is genotoxic as expressed by DNA single-strand breaks. DNA-OTA adducts have been detected in various mouse organs after ochratoxin A treatment. Tumorous tissues from three kidneys and five bladders of Bulgarian patients undergoing surgery for cancer and from three non-malignant kidneys collected from French subjects were analysed for DNA adducts. Several adducts with the same RF values as those obtained from mouse kidney after treatment with OTA (one major and some minor adducts) were detected, mainly in kidney but also in bladder tissues from Bulgaria. No adducts were detected in French kidney tissues. These results provide new evidence of the possible role of OTA in the development of tumours of the urinary tract in Bulgaria.

Animals↗
PubMed1993

Analysis of DNA adducts in smokers' lung and urothelium by 32P-postlabelling: metabolic phenotype dependence and comparisons with other exposure markers.

Carcinogen-DNA adduct levels in lung parenchyma (surgical specimens) and urothelial (exfoliated) cells of smokers, ex-smokers and non-smokers were investigated. DNA adducts were analysed by 32P-postlabelling and levels were compared with tissue-specific activity of cytochrome P450-related enzymes, or whenever possible, with metabolic phenotypes and other macromolecular adducts. Lung cancer patients who were recent smokers had significantly induced benzo[a]pyrene (BaP)-3-hydroxylase (AHH) and ethoxycoumarin O-deethylase activities in lung parenchyma compared with smoking non-cancer patients. Pulmonary AHH activity showed a good correlation with the intensity of immunohistochemical staining for P4501A(1). In lung cancer patients from Italy and Finland who were recent smokers, lung AHH activity was positively correlated (r approximately 0.65; p < 0.001) with bulky DNA adduct levels. In some lung DNA samples from smokers, the level of BaP-diol-epoxide adducts determined by HPLC with fluorescence detection showed significant positive correlation with lung AHH activity and bulky DNA adduct levels. Molecular dosimetry studies provided evidence that aromatic amines such as 4-aminobiphenyl (ABP) in tobacco smoke are primarily responsible for bladder cancer in smokers. The N-(deoxyguanosin-8-yl)-4-ABP adduct was the major smoking-related adduct in DNA of bladder biopsies from bladder cancer patients and in the DNA of exfoliated urothelial cells of smoking volunteers. The adduct levels of ABP with haemoglobin and with deoxyguanosine in urothelial DNA (determined by 32P-postlabelling) were linearly and significantly correlated, and both were related to recent cigarette smoking. Metabolic phenotype (fast/slow N-acetylator and N-oxidizer) significantly affected the levels of ABP-haemoglobin adducts.

Aryl Hydrocarbon Hydroxylases↗
PubMed1993

Results of an interlaboratory trial of 32P-postlabelling.

An interlaboratory 32P-postlabelling trial involving 15 independent investigators was conducted. Each participant was provided with four DNA samples: 1, control DNA; 2, benzo[a]pyrene-modified DNA from mouse skin; 3, DNA from the lung of a tobacco smoker; 4, 2-acetylaminofluorene-modified DNA from mouse liver. The participants analysed the DNA samples by 32P-postlabelling using the standard procedure and using two procedures, nuclease P1 digestion and butanol extraction, to enhance the sensitivity of the assay. The qualitative and quantitative reproducibility of the results obtained by the different investigators are presented. A workshop discussion on postlabelling procedures is summarized.

2-Acetylaminofluorene↗
PubMed1993

Some safety procedures for handling 32P during postlabelling assays.

32P is a high-energy (1.7 MeV) beta-emitter. Its handling is therefore subject to regulation and very strict control. During the postlabelling procedure, numerous steps involve exposure to 32P. The main risk from exposure is through irradiation, but direct accidental contamination can occur. The various manipulation steps (ATP synthesis, labelling, chromatography, quantification) have been analysed for their contribution to potential radiation exposure. Several measures have been taken in the IARC laboratories to minimize exposure of all personnel involved, including those who handle radioactive wastes, since most of the initial radioactivity is eventually discarded. The various steps to be taken for minimizing exposure, such as the training of personnel using fluorescent compounds instead of radioactivity, the use of protective screens and of equipment specially adapted for this work, are reviewed.

Equipment and Supplies↗
PubMed1992

An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smokers' lung: comparisons with total bulky adducts and aryl hydrocarbon hydroxylase activity.

An improved high-performance liquid chromatography/fluorometric assay has been established to quantitate the benzo(a)pyrene (BP) tetrols released after acid hydrolysis of lung DNA from lung cancer patients, so that the formation of benzo(a)pyrene diol-epoxide-DNA adducts can be measured. The r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydro-BP isolated by high-performance liquid chromatography was determined by chromatography in two different solvent systems and fluorescence spectroscopy. This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy- 7,8,9,10-tetrahydro-BP, requires 100-500 micrograms of DNA, and can measure 1 adduct/10(8) unmodified nucleotides. As this assay does not use immunoaffinity chromatography or solvent extraction, it allows a > 90% recovery of benzo(a)pyrene diol-epoxide-DNA adducts. This procedure has been tested on 13 DNA samples prepared from nontumorous lung parenchyma taken from lung cancer patients at surgery and revealed the presence of DNA adducts of the anti-benzo(a)pyrene diol-epoxide in 9 of 11 samples from smokers and in 2 of 2 ex-smokers. In only two samples from smokers the formation of adducts derived from syn-benzo(a)pyrene diol-epoxide was detected. A 15-fold variation in DNA adduct level was found in 11 of 13 DNA samples, with a range of 0.6-9.9 adducts of benzo(a)pyrene diol-epoxide/10(8) nucleotides. In samples containing both anti- and syn-benzo(a)pyrene diolepoxide-DNA adducts, the anti/syn adduct ratio is 2:1. A highly significant correlation was found between pulmonary microsomal aryl hydrocarbon hydroxylase activity and the level of benzo(a)pyrene diolepoxide-DNA adduct (r = 0.91; P < 0.001; n = 13). A crude linear correlation between the amounts of these adducts and those of bulky DNA adducts determined by 32P-postlabeling assay was observed in the same samples (r = 0.78; P < 0.02; n = 13). Thus this highly sensitive and specific procedure is suitable for measuring benzo(a)pyrene diolepoxide-DNA adducts in human tissues from environmentally exposed subjects and could be adapted to measure polycyclic aromatic hydrocarbons other than BP.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

Expression of pulmonary cytochrome P4501A1 and carcinogen DNA adduct formation in high risk subjects for tobacco-related lung cancer.

Cigarette smoking is the strongest risk factor for lung cancer (LC), but genetically determined variations in pulmonary metabolism of tobacco-derived carcinogens may affect individual risk. Results from a case-control study on LC patients demonstrated the pronounced effect of tobacco smoke on pulmonary xenobiotic metabolism and prooxidant state, and suggested the existence of a metabolic phenotype at higher risk for tobacco-associated LC: LC patients who were recent smokers had significantly induced BP-3-hydroxylase (AHH) and ethoxycoumarin O-deethylase (ECDE) activities in lung parenchyma, when compared with smoking non-cancer patients. In recent smokers, lung AHH activity was positively correlated with the level of tobacco smoke-derived DNA adducts as determined by 32P-postlabelling. Pulmonary AHH activity also showed a good correlation with the intensity of immunohistochemical staining for cyt. P4501A by a monoclonal Ab in lung tissue sections: smoking and peripheral type of lung cancers were positively related to high levels of this cyt. P450 species, probably reflecting high rates of induction. These results suggest that high pulmonary CYP1A1 expression (controlling in part carcinogen DNA-adduct formation) in tobacco smokers, appears to be associated with LC risk. High risk subjects may thus be identifiable through genotyping assays for CYP1A1 polymorphism.

Aryl Hydrocarbon Hydroxylases↗
PubMed1992

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.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1991

Genotoxic activity of potassium permanganate in acidic solutions.

Potassium permanganate (KMnO4) combined with sulfuric acid is a strongly oxidizing mixture which has been recommended for the destruction and the decontamination of various mutagens/carcinogens in the publication series of the International Agency for Research on Cancer. Evaluation of the genotoxicity of 4 potassium permanganate solutions was performed using a microtechnique of the Ames test with the tester strains TA97, TA98, TA100 and TA102 with and without metabolic activation. Presence of direct-acting mutagens was detected in all the samples with the tester strain TA102 without S9 mix (163-357 revertants/microliters of the solutions). Three samples containing either acetone or ethanol as an organic solvent also induced a mutagenic response on tester strain TA100 without S9 mix (167-337 revertants/microliters). In addition, DNA damage in human peripheral blood lymphocytes was also measured for one of the mixtures by a new technique: the single-cell gel assay (SCGA). A sample with no organic solvent induced DNA damage in human lymphocytes with a dose-response relationship as determined by SCGA. The major mutagenic agent generated by the permanganate solutions was found to be manganese ion (Mn2+). Both manganese sulfate (MnSO4) and manganese chloride (MnCl2) gave mutagenic dose-response relationships on tester strain TA102 without S9 mix. The mutagenic potencies were 2.8 and 2.4 revertant/nmole for MnSO4 and MnCl2 respectively. MnCl2 also induced DNA damage in human lymphocytes as determined by the SCGA. The genotoxic effects of KMnO4 in acidic conditions were probably mediated by the conversion of MnO4- to Mn2+. KMnO4 in alkaline solutions did not produce mutagenic species and may offer an alternative for the degradation of genotoxic compounds.

Biotransformation↗
PubMed1991

32P Postlabelling analysis of urinary mutagens from smokers of black tobacco implicates 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as a major DNA-damaging agent.

When mutagens extracted from the urine of two smokers of black tobacco were reacted with DNA in vitro in the presence of a metabolic activation system, several DNA adducts were detected by 32P-postlabelling analysis. Some of these adducts were also visible, but only faintly, on the autoradiogram for a non-smoker's urine. DNA adducts produced in vitro by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or 2-amino-1-methyl-6-phenylimidazo[3,5-b]pyridine could not account for the adduct pattern produced by the urinary mutagens. However, three or four 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-related DNA adducts were present among the five or six adducts observed for smokers in the autoradiograms of urinary mutagen-adducted nucleotides. Mutagenicity testing combined with HPLC fractionation of urinary extracts also supported the postlabelling data which implicates PhIP as a mutagen in the urine of smokers of black tobacco.

Chromatography, High Pressure Liquid↗
PubMed1991

Comparison of pulmonary DNA adduct levels, measured by 32P-postlabelling and aryl hydrocarbon hydroxylase activity in lung parenchyma of smokers and ex-smokers.

In order to compare pulmonary DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity, we have measured these two parameters in non-neoplastic surgical lung parenchymal samples from four ex-smokers and 19 smokers, out of 20 patients operated for lung cancer, and three for nonmalignant lung diseases. DNA adducts were determined by scintillation counting after 32P-postlabelling analysis. The microsomal fractions of the same lung specimen were assayed for AHH activity by a fluorometric method. Autoradiograms of DNA adducts found in lungs of smokers revealed two distinct diagonal radioactive zones that were absent in ex-smokers. The smokers had significantly higher levels (1.68-13.4 DNA adducts/10(8) nucleotides; mean +/- SD 5.38 +/- 3.19) than ex-smokers (0.23-2.21; 1.09 +/- 0.84). AHH activity in smokers ranged from 0.01 to 0.69 pmol/min/mg. This activity was significantly (P less than 0.05) higher in smokers (0.26 +/- 0.26) who had smoked until 1 week before surgery than in those who had stopped smoking for greater than 7 days (0.11 +/- 0.11). A positive linear correlation between DNA adduct levels and AHH activity (r = 0.69; P less than 0.001; n = 19) was found in smokers. This relationship could explain why AHH inducibility appears to be a crude marker for lung cancer risk in smokers.

Adult↗
PubMed1991

Ochratoxin A in human blood in relation to Balkan endemic nephropathy and urinary tract tumours in Bulgaria.

Ochratoxin A is suspected of being one of the etiological agents responsible for Balkan endemic nephropathy and the associated urinary tract tumours. Contamination of cereals by this mycotoxin has been found to be more frequent in areas of endemic nephropathy than in areas where the disease is absent. As ochratoxin A binds to serum albumin, it should be detectable in biological fluids from exposed populations. A survey was thus conducted to determine the occurrence of ochratoxin A in blood from people living in the endemic area who were either affected or unaffected by the two diseases and in blood from people living in control regions where these diseases do not occur. Blood samples were collected in 1984, 1986, 1989 and 1990. Ochratoxin A was found more frequently and at higher levels in blood from patients with Balkan endemic nephropathy and/or urinary tract tumours than in blood from unaffected people from endemic and control areas. These findings suggest further that ochratoxin A is involved in the etiology of the two diseases.

Balkan Nephropathy↗