In vitro anti-angiogenic effects of Hemidesmus indicus in hypoxic and normoxic conditions.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for many purposes and poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. In the context of anticancer pharmacology, anti-angiogenic therapy has become an effective strategy for inhibiting new vessel formation and contrast tumor growth. These considerations are supported by the evidence that most tumors originate in hypoxic conditions and limitation of oxygen diffusion stimulates the formation of tumor abnormal microvasculature. Aim of this study was to evaluate the in vitro anti-angiogenic potential of Hemidesmus indicus (0.31-0.93 mg/mL) on human umbilical vein endothelial cells and delineate the main molecular mechanisms involved in its anti-angiogenic activity both in normoxia and hypoxia. MATERIALS AND METHODS: The decoction of Hemidesmus indicus was subjected to an extensive HPLC phytochemical characterization. Its in vitro anti-angiogenic potential was investigated in normoxia and hypoxia. Cell proliferation, apoptosis induction, and inhibition of endothelial cell migration and invasion were analyzed by flow cytometry. The endothelial tube formation assay was evaluated in matrix gel. The capillary tube branch points formed were counted using a Motic AE21 microscope and a VisiCam videocamera. The regulation of key factors of the neovascularization process such as VEGF, HIF-1α and VEGFR-2 was explored at mRNA and protein level by real time PCR and flow cytometry, respectively. RESULTS: Treatment with Hemidesmus resulted in a significant inhibition of cell proliferation and tube formation in both normoxia and hypoxia. Hemidesmus differently regulated multiple molecular targets related to angiogenesis according to oxygen availability. In normoxia, the inhibition of VEGF was the main responsible for its anti-angiogenic effect; the angiogenesis inhibition induced in hypoxia was regulated by a more complex mechanism involving firstly HIF-1α inhibition, and then VEGF and VEGFR-2 down-regulation. Additionally, the inhibition of endothelial cell migration and invasion by Hemidesmus was more pronounced in normoxia than in hypoxia, possibly due to the physiological enhanced induction of invasion characteristic of hypoxia. CONCLUSIONS: Our results indicate that Hemidesmus might represent a promising therapeutic strategy for diseases in which the inhibition of angiogenesis could be beneficial, such as cancer. The antiangiogenic activity of Hemidesmus is based on multiple interactions with critical steps in the angiogenic cascade. VEGF expression stimulated by HIF-1α as well as endothelial cell migration and differentiation represent important targets of Hemidesmus action and might contribute to its cancer therapeutic efficacy that is presently emerging and offer a scientific basis for its use in traditional medicine.

The c.480C>G polymorphism of hOCT1 influences imatinib clearance in patients affected by chronic myeloid leukemia.

The aim of the study was to investigate any possible influence of polymorphisms of transmembrane transporters human organic cation transporter 1 (hOCT1), ABCB1, ABCG2 on imatinib pharmacokinetics in 33 men and 27 women (median age and range, 56 and 27-79 years, respectively) affected by chronic myeloid leukemia. A population pharmacokinetic analysis was performed to investigate imatinib disposition in every patient and the role of transporter polymorphisms. Results showed that the α1-acid glycoprotein and the c.480C>G genotype of hOCT1 had a significant effect on apparent drug clearance (CL/F) being responsible, respectively, for a 20% and 10% decrease in interindividual variability (IIV) of CL/F (from 50.1 up to 19.6%). Interestingly, 25 patients carrying at least one polymorphic c.480 G allele had a significant lower CL/F value with respect to the 35 c.480CC individuals (mean±s.d., 9.6±1.6 vs 12.1±2.3 l h(-1), respectively; P<0.001). In conclusion, the hOCT1 c.480C>G SNP may significantly influence imatinib pharmacokinetics, supporting further analyses in larger groups of patients.

Hemidesmus indicus induces apoptosis as well as differentiation in a human promyelocytic leukemic cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever, and diarrhea. Poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. The aim of this study was to investigate the cytodifferentiative, cytostatic and cytotoxic potential of a decoction of Hemidesmus indicus's roots (0.31-3 mg/mL) on a human promyelocytic leukemia cell line (HL-60). MATERIALS AND METHODS: The decoction of Hemidesmus indicus was characterized by HPLC to quantify its main phytomarkers. Induction of apoptosis, cell-cycle analysis, levels of specific membrane differentiation markers were evaluated by flow cytometry. The analysis of cell differentiation by nitroblue tetrazolium (NBT) reducing activity, adherence to the plastic substrate, α-napthyl acetate esterase activity and morphological analysis was performed through light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Starting from the concentration of 0.31 mg/ml, Hemidesmus indicus induced cytotoxicity and altered cell-cycle progression, through a block in the G0/G1 phase. The decoction caused differentiation of HL-60 cells as shown by NBT reducing activity, adherence to the plastic substrate, α-naphtyl acetate esterase activity, and increasing expression of CD14 and CD15. The morphological analysis by LM and TEM clearly showed the presence of granulocytes and macrophages after Hemidesmus indicus treatment. CONCLUSIONS: The cytodifferentiating, cytotoxic and cytostatic activities of Hemidesmus indicus offers a scientific basis for its use in traditional medicine. Its potent antileukemic activity provides a pre-clinical evidence for its traditional use in anticancer pharmacology. Further experiments are worthwhile to determine the in vivo anticancer potential of this plant decoction and its components.

Guanosine protects human neuroblastoma cells from oxidative stress and toxicity induced by Amyloid-beta peptide oligomers.

Amyloid-beta (Abeta) peptide aggregation forms such as soluble oligomers (O) have a causal role in neuronal dysfunction and death associated with Alzheimer?s Disease (AD). The main efforts for the development of neuroprotective drugs are therefore focused on preventing Abeta production, aggregation or downstream neurotoxic events. We therefore investigated the effect of guanosine (GUO), a guanine based purine, that exerts neurotrophic and neuroprotective effects. The GUO showed the ability to reduce neuronal death in terms of apoptosis, but not necrosis, elicited by Abeta1-42O in human neuroblastoma SH-SY5Y cells. The neuroprotective effect was recorded only when the GUO was added simultaneously to treatment of the SH-SY5Y cells with Abeta1-42O. By contrast, the GUO treatment of SH-SY5Y cells before and after the appearance of beta1-42O toxicity had no neuroprotective effects. The employment of specific inhibitors showed the involvement of neuronal survival pathways, such as PI3K?Akt and MAPK-ERK for the GUO anti-apoptotic effects observed. In parallel, the SH-SY5Y cells treated with GUO, in experimental conditions similar to those adopted to evaluate neuronal death, showed a marked decrease of the early reactive oxygen species formation induced by Abeta1-42O and pro-oxidant H2O2. In the same neuronal model, GUO was also shown to inhibit the extra- and intra-cellular Abeta1-42 release as well as the beta-secretase activity evoked by H2O2 pro-oxidant action. Based on these findings, GUO and other guanine based purines appear to be a promising class of compounds with neuroprotective properties that may play an important role in the therapy of AD.

Influence of cultivation system on bioactive molecules synthesis in strawberries: spin-off on antioxidant and antiproliferative activity.

Strawberries (Fragaria ananassa L., cv. favette) were studied to investigate the influence of cultivation practices (biodynamic, conventional) on the synthesis of bioactive molecules (ascorbic acid, ellagic acid, anthocyanins, flavonols) and to evaluate their antioxidant activity. Additionally, the in vitro bioactivity, in terms of antioxidant and antiproliferative activity, of the same strawberry samples in human colon carcinoma (Caco-2) cells was also studied. Compared to conventional strawberries, biodynamic fruits had a significantly higher content of ascorbic acid (P < 0.01), pelargonidin-3-glucoside (P < 0.05), cyanidin-3-glucoside (P < 0.01), ellagic acid (P < 0.01), quercetin, and kaempferol (both P < 0.01). Antioxidant activity of biodynamic strawberry crude extract was significantly higher than that of the conventional one (P < 0.05); in addition, while the antioxidant activity of water-soluble fraction was very similar in both biodynamic and conventional strawberries, that of water-insoluble fraction of biodynamic fruits was significantly higher (P < 0.05). The same crude extract of biodynamic strawberry samples effectively corresponded to an increase of bioactivity, in terms of both cellular antioxidant activity and antiproliferative activity, in Caco-2 cells differentiated to normal intestinal epithelia and in undifferentiated Caco-2, respectively. Further studies are needed to confirm whether the practice of biodynamic agriculture is likely to increase the bioactivity of other varieties of fruits and vegetables.

Drinking water quality: an in vitro approach for the assessment of cytotoxic and genotoxic load in water sampled along distribution system.

An in vitro approach was performed to assess the quality of drinking water collected at two treatment/distribution networks located near the source (Plant #1) and the mouth of River Po (Plant #2). The water was sampled at different points of each distribution network, before (raw water) and after the chlorine dioxide disinfection, and in two points of the pipeline system to evaluate the influence of the distribution system on the amount and quality of the disinfection by-product. Cytotoxicity and genotoxicity of water extracts were evaluated in human peripheral lymphocytes and Hep-G2 cells by the use of the micronucleus (MN) test and Comet assay. Raw water samples of both plants induced cytotoxic effects, but not the increases of MN frequency in Hep-G2 cells and in human lymphocytes. Increases of DNA damage in human leukocytes was detected by Comet assay for raw water of Plant #2 at concentration > or = 0.25 Leq/mL. The disinfection process generally has reduced the toxicity of water samples, even if potential direct DNA-damaging compounds have been detectable in drinking water samples. The proposal approach, if currently used together with chemical analysis, can contribute to improve the monitoring drinking water.

Interaction of the isothiocyanate sulforaphane with drug disposition and metabolism: pharmacological and toxicological implications.

Isothiocyanates (ITCs) are sulfur-containing compounds that are broadly distributed among cruciferous vegetables such as cabbages and broccoli. The consumption of ITCs is expected to rise due to the use of dietary supplements and public health initiatives promoting the consumption of more fruits and vegetables. Sulforaphane (SFN) is by far the most widely studied and characterized ITC. SFN is extensively metabolized and can therefore compete with other substrates of Phases I, II, III enzymes and transporters. In addition, it has an unusually high potency as an inducer of phase II enzymes and regulates the expression and function of different cytochrome P-450 genes. Such effects can be beneficial and may indicate a mechanism for the preventive role that SFN is believed to play against the degenerative events of aging and chronic diseases. Furthermore, these gene induction effects and the interaction with detoxification responses can modify bioavailability and in vivo bioactivity of drugs. This review will discuss 1) the metabolism of ITCs using SFN as an example, 2) inhibition of drug metabolism by SFN, and 3) induction of drug metabolizing enzymes by SFN. The potential pharmacological and toxicological implications of these effects on drug metabolism will also be discussed.

Chemoprevention of cancer by isothiocyanates and anthocyanins: mechanisms of action and structure-activity relationship.

Carcinogenesis is a multi-step, multi-path and multi-focal process, which involves a series of epigenetic and genetic alterations that begin with genomic instability and end with the development of cancer. This long and complex process presents opportunities for the development of interventions both in preventing cancer initiation and in treating the neoplasm during its premalignant stages. Failure and high systemic toxicity of conventional cancer therapies have accelerated the search for newer agents, which could prevent and/or slow-down cancer growth and have more human acceptability by being less or non-toxic. Now, it is recognized that diets rich in fruits and vegetables are associated with lower risk of cancer. Taking cue from these observations, there is a strong interest in isolating and characterizing the nutritive and non-nutritive components of fruits and vegetables as potential chemopreventive agents. Isothiocyanates and anthocyanins, present in widely consumed fruits and vegetables, are two such agents. In recent years, increasing body of evidence has underscored the cancer preventive efficacy of isothiocyanates and anthocyanins in both in vitro and in vivo animal models. In this review article, we will provide detailed insight into the chemopreventive efficacy of isothiocyanates and anthocyanins based on the evidence generated from various studies performed using cell culture or animal models of epithelial cancers. Moreover, we will discuss the potential clinical relevance of the observed chemopreventive effects of these agents.

Role of quercetin in modulating rat cardiomyocyte gene expression profile.

Despite extensive studies, the fundamental mechanisms responsible for the development and progression of cardiovascular diseases have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species play a major pathological role. Oxidative stress reduction induced by flavonoids has been regarded by many as the most likely mechanism in the protective effects of these compounds; however, there is an emerging view that flavonoids may also exert modulatory actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid present in the human diet, has been widely studied, and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. The main goal of this study was to examine the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin using DNA microarrays and to relate these data to functional effects. Results showed distinct temporal changes in gene expression induced by quercetin and a strong upregulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect antioxidant mechanism.

Antioxidant effectiveness of organically and non-organically grown red oranges in cell culture systems.

BACKGROUND: Consumers consider plant food products from organic origin healthier than the corresponding conventional plant foods. Clear experimental evidence supporting this assumption is still lacking. AIM OF THE STUDY: To determine if the organic red oranges have a higher phyto-chemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and in vitro bioactivity, in terms of protective effect against oxidative damage at cellular level, than nonorganic red oranges. METHODS: Total phenolics were measured using the Folin Ciocalteau assay, while total anthocyanins and ascorbic acid levels were determined by spectrophotometric and HPLC analysis, respectively. In addition, the total antioxidant activity of red orange extracts was measured by the ABTS(*+) test. The ability of red orange extracts to counteract conjugated diene containing lipids and free radical production in cultured rat cardiomyocytes and differentiated Caco-2 cells, respectively, was assessed. RESULTS: Organic oranges had significantly higher total phenolics, total anthocyanins and ascorbic acid levels than the corresponding non-organic oranges (all p < 0.05). Moreover, the organic orange extracts had a higher total antioxidant activity than non-organic orange extracts (p < 0.05). In addition, our results indicate that red oranges have a strong capacity of inhibiting the production of conjugated diene containing lipids and free radicals in rat cardiomyocytes and differentiated Caco-2 cells, respectively. Statistically higher levels of antioxidant activity in both cell models were found in organically grown oranges as compared to those produced by integrated agriculture practice. CONCLUSIONS: Our results clearly show that organic red oranges have a higher phytochemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and bioactivity than integrated red oranges. Further studies are needed to confirm whether the organic agriculture practice is likely to increase the antioxidant activity of other varieties of fruits and vegetables.

A mixture of isothiocyanates induces cyclin B1- and p53-mediated cell-cycle arrest and apoptosis of human T lymphoblastoid cells.

As with other candidate chemopreventive agents, most of our knowledge on the biological effects of isothiocyanates (the many sulfur-containing metabolites found in cruciferous vegetables) comes from studies of single natural or synthetic compounds. To investigate whether the biological/chemopreventive effects of administration of single isothiocyanates can differ from those of a mixture of isothiocyanates, we tested the effects of a mixture of four different isothiocyanates on cell-cycle progression and apoptosis in human T leukemia Jurkat cells, and identified some of the molecular pathways triggered by the mixture. The mixture affected critical points of the cell cycle via modulation of the expression of cyclin B1. Moreover, it induced apoptosis, mediated by an increase in p53 and bax (expression of bcl-2 was unaffected). Comparison of the data with those previously obtained with the single isothiocyanates under identical experimental conditions provides evidence that the quantitative effects of a single, specific isothiocyanate can be significantly different from those of an isothiocyanate mixture at realistic doses.

[Biomarkers of effects and susceptibility for risk assessment]. / Biomarcatori di effetto e di suscettibilità per la valutazione del rischio.

The ambient air of urban centres is polluted with potentially toxic chemicals mostly arising from the combustion or fuels used for transport, heating and industrial activities. Alongside the risk to the general public, atmospheric pollution could be considered an occupational health hazard to professional groups, such us traffic police or professional drivers working in urban areas. Molecular epidemiology can facilitate health risk assessment by investigating the relationship between exposure to environmental pollutants and quantification of biomarkers that lie on the pathway of carcinogenesis upstream of clinical disease. In particularly, biomarkers of early effects and susceptibility are playing an increase role in the investigation of the impact of air pollution on human carcinogenesis.

Photomutagenic properties of terfenadine as revealed by a stepwise photostability, phototoxicity and photomutagenicity testing approach.

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm(-2) UVA-UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm(-2) UVA-UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity--carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

Mutagenic and clastogenic activity of gastric juice in human gastric diseases.

To investigate whether subjects with low-acid states are exposed to increased genetic risk with respect to controls, we evaluated mutagenicity and presence of clastogenic factors (CF) in the gastric juice of chronic atrophic gastritis and omeprazole-treated patients. Mutagenic gastric juice was found in 8/15 (53%) chronic atrophic gastritis patients, 8/11 (73%) omeprazole-treated patients, and 2/13 (15%) healthy control subjects. The mean mutagenicity ratio of omeprazole-treated patients (1.52+/-0.48/0.1 ml gastric juice) was significantly higher than those of either controls (1.07+/-0.15; P<0.01) or chronic atrophic gastritis patients (1.16+/-0.21; P<0.05). Only chronic atrophic gastritis patients showed an increased clastogenic index with respect to healthy controls (2.67+/-2.13 versus 0.38+/-0.51; P<0.001). These findings expand our knowledge of gastric disease risk factors, and indicate that there may well be a risk of mucosal DNA damage arising from the presence of mutagenic and CF in the gastric juice.

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes.

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.

Micronuclei induction, cell cycle delay and apoptosis as markers of cellular stress caused by ursodeoxycholic acid in human lymphocytes.

Ursodeoxycholic acid (UDCA) is a bile acid (BA) used for cholesterol gallstone dissolution. Since epidemiological evidence indicates that BAs can be involved in the etiology of colorectal cancer, we investigated the effects of UDCA and its physiologically produced taurine conjugate tauroursodeoxycholic acid (TUDCA) on human lymphocyte cultures in terms of genetic damage in the form of micronuclei (MN) production, cell cycle modifications and induction of apoptosis. With respect to controls, treatment with UDCA (from 10 microg/ml) caused a dose-related increase in MN, whereas TUDCA caused no significant increase (up to 1000 microg/ml). Fluorescence in situ hybridization (FISH) analysis using pancentromeric probes suggested that UDCA exerts aneugenic activity. Bromodeoxyuridine/Hoechst flow cytometry showed that both BA significantly inhibit cell cycle progression (UDCA at 100 microg/ml, and TUDCA, more markedly at 300-1000 microg/ml). Neither UDCA nor TUDCA affected induction of apoptosis, as evaluated by the Annexin-V-Fluos assay. We conclude that UDCA is potentially genotoxic. However, taking into account the characteristics of other physiological BA, our findings are in line with the concept that long-term UDCA treatment may be safely administered. The multi-assay approach reported here could be useful in the toxicological evaluation of newly developed BA analogs as candidates for pharmacological use.

Photostability and phototoxicity studies on diltiazem.

The photostability of diltiazem was studied in aqueous solutions at different pH values. Firstly, the hydrolysis of the drug to desacetyldiltiazem in alkaline medium was evaluated and then the drug photodegradation under exposure to UVA-UVB radiation (solar simulator) was monitored by HPLC methods. The main photoproduct was isolated and characterized as diltiazem-S-oxide on the basis of the NMR and mass spectra. The HPLC method was also applied to the selective analysis of diltiazem in commercial formulations. Tests on mutagenicity and photomutagenicity of the drug were also carried out using Salmonella typhimurium TA 102 strain. In this testing the drug neither was mutagenic nor toxic.

Studies on the photostability and in vitro phototoxicity of Labetalol.

The purpose of this study was to obtain information on the photochemical and phototoxic properties of Labetalol, a beta-blocker drug. Preliminary information on the drug photoreactivity was achieved using a flow system with a photochemical reactor on-line with a diode array detection system. Photophysical and photochemical investigations on the drug were performed in aqueous solutions at different pH values using spectrophotometric and fluorimetric methods; the photodegradation quantum yield was found to be 2.7 x 10(-3) at pH 5.8 and 1.5 x 10(-2) at pH 11.5. Forced photodegradation of labetalol solutions under exposure to UVA--UVB radiations (xenon arc lamp) was monitored by reversed-phase liquid chromatography. The main photodegradation products were isolated and characterized by NMR and mass spectrometry; labetalol was found to give 3-amino-1-phenylbutane and salicylamide-4-carboxaldehyde as the main photoproducts. Preliminary phototoxic testings on human keratinocyte cultures were performed evaluating the viability of the cells by the neutral-red uptake assay; mutagenic and photomutagenicity tests were also carried out based on Salmonella typhimurium strains. As a result, labetalol was found to be photolabile,mainly in alkaline medium, but evidences of significant phototoxic and photomutagenic effects by the drug were not observed.

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei.

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

Increased cytogenetic damage detected by FISH analysis on micronuclei in peripheral lymphocytes from alcoholics.

Alcohol abuse greatly increases the risk of various malignancies, including cancer of the liver and digestive tract. Although it is thought that this may be due, at least partially, to the mutagenic properties of ethanol, little is known about the genotoxic effects of ethanol in humans. We investigated the chromosomal damage in lymphocytes from 20 alcoholics and 20 controls using the micronucleus (MN) assay combined with fluorescence in situ hybridization (FISH) with a pancentromeric DNA probe capable of differentiating centromere positive (C+) from centromere negative (C-) MN. The frequency of MN in binucleate lymphocytes was significantly higher in alcoholics than in controls (12.0 +/- 5.4 and 7.6 +/- 1.6, respectively; P: < 0.05). FISH revealed significantly higher frequencies of C+ MN in alcoholics than in controls (8.2 +/- 4.8 and 3.4 +/- 1.4, respectively; P: < 0.05). In the alcoholics, no association was found between years of alcohol abuse and frequency of MN or C+ MN. However, age influenced MN and C+ MN frequency both in alcoholics and controls. These results indicate that alcohol abuse may well induce chromosome loss in humans, suggesting a possible aneugenic mechanism of alcohol. This effect could contribute to the health hazards related to alcoholism such as cancer risk.