Characterization of a melamino nitroheterocycle as a potential lead for the treatment of human african trypanosomiasis.

This paper reports an evaluation of a melamino nitroheterocycle, a potential lead for further development as an agent against human African trypanosomiasis (HAT). Studies on its efficacy, physicochemical and biopharmaceutical properties, and potential for toxicity are described. The compound previously had been shown to possess exceptional activity against Trypanosoma brucei in in vitro assays comparable to that of melarsoprol. Here, we demonstrate that the compound also was curative in the stringent acute mouse model T. brucei rhodesiense STIB 900 when given intraperitoneally at 40 mg/kg of body weight. Nevertheless, activity was only moderate when the oral route was used, and no cure was obtained when the compound was tested in a stage 2 rodent model of infection. Genotoxic profiling revealed that the compound induces DNA damage by a mechanism apparently independent from nitroreduction and involving the introduction of base pair substitutions (Ames test), possibly caused by oxidative damage of the DNA (comet test). No significant genotoxicity was observed at the chromosome level (micronucleus assay). The lack of suitable properties for oral and central nervous system uptake and the genotoxic liabilities prevent the progression of this melamine nitroheterocycle as a drug candidate for HAT. Further modification of the compound is required to improve the pharmacokinetic properties of the molecule and to separate the trypanocidal activity from the toxic potential.

Multicentre study for the evaluation of mutagenic/carcinogenic risk in nurses exposed to antineoplastic drugs: assessment of DNA damage.

OBJECTIVES: People who handle antineoplastic drugs, many of which classified as human carcinogens by International Agency for Research on Cancer, are exposed to low doses in comparison with patients; however, the long duration of exposure could lead to health effects. The aim of this work was to evaluate DNA damage in white blood cells from 63 nurses who handle antineoplastic drugs in five Italian hospitals and 74 control participants, using different versions of the Comet assay. METHODS: Primary DNA damage was assessed by using the alkaline version of the assay on leucocytes, whereas to detect DNA oxidative damage and cryptic lesions specifically, the Comet/ENDO III assay and the Comet/araC assay were performed on leucocytes and lymphocytes, respectively. RESULTS: In the present study, no significant DNA damage was correlated with the work shift. The exposed population did not differ significantly from the reference group with respect to DNA primary and oxidative damage in leucocytes. Strikingly, in isolated lymphocytes treated with araC, lower data dispersion as well as a significantly lower mean value for the percentage of DNA in the comet tail was observed in exposed participants as compared with the control group (p<0.05), suggesting a potential chronic exposure to crosslinking antineoplastic drugs. CONCLUSIONS: Although stringent rules were adopted at national and international levels to prevent occupational exposure to antineoplastic drugs, data reported in this study support the idea that a more efficient survey on long-lasting exposures at very low concentrations is needed.

A study protocol for the evaluation of occupational mutagenic/carcinogenic risks in subjects exposed to antineoplastic drugs: a multicentric project.

BACKGROUND: Some industrial hygiene studies have assessed occupational exposure to antineoplastic drugs; other epidemiological investigations have detected various toxicological effects in exposure groups labeled with the job title. In no research has the same population been studied both environmentally and epidemiologically. The protocol of the epidemiological study presented here uses an integrated environmental and biological monitoring approach. The aim is to assess in hospital nurses preparing and/or administering therapy to cancer patients the current level of occupational exposure to antineoplastic drugs, DNA and chromosome damage as cancer predictive effects, and the association between the two. METHODS/DESIGN: About 80 healthy non-smoking female nurses, who job it is to prepare or handle antineoplastic drugs, and a reference group of about 80 healthy non-smoking female nurses not occupationally exposed to chemicals will be examined simultaneously in a cross-sectional study. All the workers will be recruited from five hospitals in northern and central Italy after their informed consent has been obtained.Evaluation of surface contamination and dermal exposure to antineoplastic drugs will be assessed by determining cyclophosphamide on selected surfaces (wipes) and on the exposed nurses' clothes (pads). The concentration of unmetabolized cyclophosphamide as a biomarker of internal dose will be measured in end-shift urine samples from exposed nurses. Biomarkers of effect and susceptibility will be assessed in exposed and unexposed nurses: urinary concentration of 8-hydroxy-2-deoxyguanosine; DNA damage detected using the single-cell microgel electrophoresis (comet) assay in peripheral white blood cells; micronuclei and chromosome aberrations in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e. glutathione S-transferases) will also be analysed.Using standardized questionnaires, occupational exposure will be determined in exposed nurses only, whereas potential confounders (medicine consumption, lifestyle habits, diet and other non-occupational exposures) will be assessed in both groups of hospital workers.Statistical analysis will be performed to ascertain the association between occupational exposure to antineoplastic drugs and biomarkers of DNA and chromosome damage, after taking into account the effects of individual genetic susceptibility, and the presence of confounding exposures. DISCUSSION: The findings of the study will be useful in updating prevention procedures for handling antineoplastic drugs.

An Observational Study on Acupuncture for Earthquake-Related Post-Traumatic Stress Disorder: The Experience of the Lombard Association of Medical Acupuncturists/Acupuncture in the World, in Amatrice, Central Italy.

Background and Objective: Earthquakes are associated with severe psychiatric disorders, such as anxiety, depression, and post-traumatic stress disorder (PTSD). Current first-line therapies for PTSD have well-known side-effects. Acupuncture is a complementary approach to help patients cope with mental problems after natural disasters and public health events. This article describes an acupuncture intervention conducted by the Lombard Association of Medical Acupuncturists/Acupuncture in the World in the earthquake-stricken area of Amatrice in Central Italy and measures the effect of acupuncture on earthquake-related pain and psychologic symptoms in the victims. Methods: The intervention lasted 5 weeks, from September to October 2016. Adult patients with psychologic symptoms and musculoskeletal pain were included. Treatments were performed by experienced medical acupuncturists. A verbal/numerical scale was developed to quantify the effect of intervention. A Wilcoxon rank-sum test was used for comparison of the scores before and after the acupuncture treatment. Results: Of the patients, 68.3% reported having both pain and psychologic symptoms. The most frequently used meridian points were Kidney (13.17%), followed by Large Intestine (12.46%), Spleen (12.04%) and Gall Bladder (10.34%). After 3 treatments performed in daily sessions, 54.05% and 60.6% of patients reported marked improvements in psychologic and pain symptoms, respectively. Statistical analysis showed a significant difference between the scores reported before the first treatment and after the third treatment, both for pain (P = 0.000) and psychologic symptoms (P = 0.000). No serious adverse events were reported. Conclusions: These results suggest that acupuncture could be a useful tool for reducing pain and psychologic symptoms related to earthquakes, but further research is required in this specific area.

Cytotoxic and genotoxic potential of drinking water: a comparison between two different concentration methods.

The level of exposure to hazardous compounds through drinking water is low but it is maintained throughout life, therefore representing a risk factor for human health. The use of techniques averaging the consumer's exposure over time could be more useful than relying on intermittent grab samples that may misrepresent average tap water concentrations due to short-term temporal variability. In this study, we compared the induction of in vitro cytotoxic and genotoxic effects (DNA damage by the comet assay) in relation to different sampling methods, i.e. exposure over time (semipermeable membrane devices, SPMDs, exposed for 30 days) or intermittent grab samples (5 weekly water sampling, C18 concentration). Waters with different chemical characteristics were sampled to test the sensitivity of the two methods. We did not found any positive correlation between the biological findings and water chemical parameters. SPMD extracts induced a significantly greater DNA damage than C18. The different behaviour was specially found for the water samples with a low level of organic compounds and when C18 extracts were highly cytotoxic. Our findings suggest that SPMD could be of a great interest in assessing genotoxic contaminants in both raw and drinking water, with great suitability for continuous monitoring. Furthermore, the results of this study have confirmed the great importance of the biological assays in evaluating the effects of a complex mixture such as water in addition to the conventional chemical examination of water quality.

Trypanocidal nitroimidazole derivatives: relationships among chemical structure and genotoxic activity.

Human American trypanosomiasis is resurgent in Latin Americans, and new drugs are urgently required as current medications suffer from a number of drawbacks. Some nitroheterocycles have been demonstrated to exert a potent activity against trypanosomes. However, host toxicity issues halted their development as trypanocides. As part of the efforts to develop new compounds in order to treat parasitic infections, it is important to define their structure-activity relationship. In this study, 5-nitromegazol and two of its analogues, 4-nitromegazol, and 1-methyl-5-nitro-2-imidazolecarboxaldehyde 5-nitroimidazole-thiosemicarbazone, were tested and compared for in vitro induction of DNA damage in human leukocytes by the comet assay, performed at different pHs to better identify the types of damage. Specific oxidatively generated damage to DNA was also measured by using the comet assay with endonucleases. DNA damage was found in 5-nitromegazol-treated cells: oxidative stress appeared as the main source of DNA damage. 4-Nitromegazol did not produce any significant effect, thus confirming that 4-nitroimidazoles isomers have no important biological activity. The 5-nitroimidazole-thiosemicarbazone induced DNA damage with a higher efficiency than 5-nitromegazol. The central role in the reduction process played by the acidic hydrazine proton present in the thiosemicarbazone group but not in the cyclic (thiadiazole) form can contribute to rationalise our results. Given its versatility, thiosemicarbazone moiety could be involved in different reactions with nitrogenous bases (nucleophilic and/or electrophilic attacks).

Evaluation of neurodevelopmental effects on rats exposed prenatally to sulfentrazone.

Although some studies have pointed to embryo/fetal toxicity at treatment levels that were not maternally toxic, knowledge about the potential toxic effects of the herbicide sulfentrazone is still limited. Since the results of these studies have raised some concern, the present work studied the effects of sulfentrazone maternal exposure on the physical and neurobehavioral endpoints in the development of rat pups. To accomplish that, the effects of the herbicide sulfentrazone (25 and 50mg/kg) were examined at two different developmental stages in rats: during the first 6 days of gestation, or in the organogenesis period (6-15 days). After parturition, pups were tested in a developmental test battery including measures of growth, maturational milestones, and neurobehavioral development. Maternal exposure to the herbicide resulted in significant alterations of the postnatal age at which the developmental milestones of ear and eye opening and testes descent were observed. There was a reduced weight gain rate in pups and their mothers when treated during the gestational period at the highest dose tested. Also, the functional state of the rat pup nervous system at different stages of postnatal development showed some neurodevelopmental delays in righting reflex, negative geotaxis, grip response, and motor coordination-locomotion and rearing (21-90 days of life) in the treated groups. Herbicide genotoxicity was investigated in fresh leukocytes both in mothers and pups using the comet assay: the data did not show any significant genotoxic effect induced by the herbicide. The findings of this study emphasize that sulfentrazone maternal exposure may lead to some neuromuscular and behavioral deficits in nursing pups.

Neurodevelopmental effects of perinatal fenarimol exposure on rats.

Knowledge about the potential toxic effects of fenarimol, a widely used fungicide, is still limited. Fenarimol is an aromatase inhibitor and therefore can affect estrogen/androgen levels in vivo in rodents. In view of these facts, the aim of the present work was to study the effects of fenarimol maternal exposure during different critical phases in the development of central nervous system in rat pups, on early physical and neurobehavioral endpoints essential to their development. For that, the effects of the fungicide fenarimol (150 and 300 mg/kg) were examined at three different developmental stages in the rat: during the first 6 days of gestation, prenatal (15-21 days), or first 6 days of lactation. Three categories of the impact of fenarimol on neonatal growth and neurobehavioral development of offspring were assessed: (1) physical, (2) reflex and strength, and (3) motor coordination. Findings on the pups' physical development did not indicate any significant alterations of the postnatal age at which specific developmental milestones were observed (pinna detachment, development of the fur, eruption of the incisor teeth, opening of the ears and eyes and testes descent). However, there was a reduced rate of weight gain in pups of mothers treated during lactation related to the earlier testing time periods (1-23 days of life). The study of the functional state of the rat pup nervous systems at different stages of postnatal development revealed some neurodevelopmental delays in righting reflex, climbing and grip response and locomotion (20-90 days of life) in the treated groups. Taken together, findings of this study emphasize that, as a result of fenarimol maternal exposure, some neuromuscular and behavioral deficits in nursing pups may occur principally during the last gestational period and lactation. These results could be the basis for further studies on molecular actions of fenarimol in order to predict better the biological consequences of this fungicide.

In vitro cytotoxicity and genotoxicity of chlorinated drinking waters sampled along the distribution system of two municipal networks.

When chlorine is used as a disinfectant for drinking water it may react with organic materials present in or released by the water pipes and thus form by-products that may represent a genotoxic hazard. The aim of this study was to assess the potential genotoxicity and cytotoxicity of extracts of chlorinated drinking water supplied by local aquifers of two Italian towns, Plants 1 and 2, located in the sub-Alpine area and on the Po plain, respectively. The raw water fell within the legal limits with regards to its chemical and physical properties. Water from Plant 2 contained higher levels of total organics (TOC) and nitrate than water from Plant 1. Water was sampled at different points along the distribution networks to evaluate the influence of the system on the amount and quality of the by-products. Cytotoxic and genotoxic damage was assessed in freshly isolated human white blood cells (WBC) and Hep-G2 cells by use of the micronucleus (MN) test and the Comet assay to measure primary DNA damage. While they did not show significant cytotoxicity, all Plant 1 water concentrates induced short-time genotoxic effects on leukocytes at concentrations > or =1 Lequiv./mL. Plant 2 samples were able to induce cytotoxic effects in both Hep-G2 cells and leukocytes. Furthermore, although there was no significant increase in MN frequency, DNA migration was strongly increased both in human leukocytes (> or =0.5 Lequiv./mL, 1h treatment, water samples collected from all points) and in Hep-G2 cells (> or =0.75 Lequiv./mL, 24 h treatment, tap water sampled at the nearest distribution point). The current use of these in vitro cytotoxicity/genotoxicity tests together with the normal chemical analyses could provide information to help water-works managers and health authorities evaluate drinking water quality and adopt strategies to reduce genotoxic compounds in tap water and prevent human exposure to these compounds.

Detection of mutagens in water-distribution systems after disinfection.

This research examined the quality of water-before and after distribution-of four drinking-water production plants located in Northern Italy, two of which collected water from local aquifers and two from the River Po. A battery of genotoxicity assays for monitoring drinking-water was performed to assess the quality of the water produced by the treatment plants under study. Three different sampling stations were selected at each plant, one right at the outlet of the treatment plant and two along with the distribution pipelines. Raw river water was also sampled and analysed as a control. The water samples (500 l) were concentrated on silica C18 cartridges and the extracts were tested in in vitro mutagenicity assays (Salmonella/microsome assay with strains TA 98 and TA 100; SOS Chromotest with Escherichia coli strain PQ37); gene conversion, point mutation and mitochondrial DNA mutability assays with the diploid Saccharomyces cerevisiae strain D7 and a toxicity test using the bioluminescent bacterium Vibrio fischeri (Microtox). The Microtox test and the mitochondrial DNA mutability assay showed the greatest sensitivity towards toxic or mutagenic substances in the water extracts considered. The results show that this battery of short-term tests is applicable in the routine monitoring of drinking-water quality before and after distribution.

A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters.

Since the 1980s, stricter water quality regulations have been promulgated in many countries throughout the world. We discuss the application of a battery of both in vivo and in vitro genotoxicity tests on lake water as a tool for a more complete assessment of surface water quality. The lake water concentrated by adsorption on C18 silica cartridges were used for the following in vitro biological assays: gene conversion, point mutation, mitochondrial DNA mutability assays on the diploid Saccharomyces cerevisiae D7 strain, with or without endogenous P450 complex induction; DNA damage on fresh human leukocytes by the comet. Toxicity testing on yeast and human cells was also performed. In vivo genotoxicity was determined by the comet assay on two well-established bio-indicator organisms of water quality (Cyprinus carpio erythrocytes and Dreissena polymorpha haemocytes) exposed in situ. The in vivo experiments and the water samplings were carried out during different campaigns to detect seasonal variations of both the water contents and physiological state of the animals. Temperature and oxygen level seasonal variations and different pollutant contents in the lake water appeared to affect the DNA migration in carp and zebra mussel cells. Seasonal variability of lake water quality was also evident in the in vitro genotoxicity and cytotoxicity tests, with regards to water pollutant quantity and quality (direct-acting compounds or indirect-acting compounds on yeast cells). However, the measured biological effects did not appear clearly related to the physical-chemical characteristics of lake waters. Therefore, together with the conventional chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs: their use could permit the quantification of mutagenic hazard in surface waters.

Use of the Comet test and micronucleus assay on human white blood cells for in vitro assessment of genotoxicity induced by different drinking water disinfection protocols.

Surface water disinfection can lead to the formation of mutagenic/carcinogenic by-products derived from reactions with naturally occurring inorganic compounds. We investigated the feasibility and potential usefulness of an integrated approach to genotoxicity analysis of drinking water. The approach employed the Comet and micronucleus (MN) assays to evaluate the DNA and chromosomal damage produced by water extracts in human blood cells. Surface water samples from Lago Trasimeno (Italy) were collected in different seasons (July 2000, October 2000, February 2001, and June 2001), and samples were disinfected with sodium hypochloride (NaClO), chlorine dioxide (ClO(2)), or peracetic acid (PAA). Extracts of untreated and treated water were incubated with primary human leukocytes. The Comet assay revealed both strong seasonal variations and differences between samples processed by the three disinfection protocols. The three disinfectants increased the genotoxicity of the water collected in July 2000 and October 2000, with PAA producing the greatest amount of DNA damage. Extracts of raw water collected in February 2001 produced so much DNA damage that the relative genotoxic potentials of the three disinfectants could not be evaluated. No increase in MN frequency was detected in any of the samples. The multi-endpoint MN assay indicated, however, that our study samples (especially the sample collected in the February 2001) were cytotoxic. We conclude that this integrated approach to genotoxicity assessment may be useful both for the quality control of raw drinking water and to help compare the potential health risks associated with alternative disinfection processes.

Modulating effects of humic acids on genotoxicity induced by water disinfectants in Cyprinus carpio.

The use of chlorinated disinfectants during drinking-water production has been shown to generate halogenated compounds as a result of interactions of humic acids with chlorine. Such chlorinated by-products have been shown to induce genotoxic effects and consumption of chlorinated drinking-water has been correlated with increased risk for cancer induction in human populations. The aim of this work was to test the potential genotoxic effects on circulating erythrocytes of the fish Cyprinus carpio exposed in vivo to well-waters disinfected with sodium hypochlorite (NaClO), chlorine dioxide (ClO2) or peracetic acid (CH3COO2H, PAA), in the absence or presence of standard humic acids (HA). The effects were measured by use of the micronucleus (MN) and the single-cell gel electrophoresis (Comet) assays at different sampling times after a 3-day exposure period. The exposure to chlorine disinfectants without the addition of HA produced a clear toxic effect. Significant cytogenetic damage (i.e. MN induction) was detected in fish populations exposed to both NaClO and ClO2 with humic acids. In the Comet assay, a significant decrease of DNA migration was observed in erythrocytes of specimens after exposure to NaClO-disinfected water without HA. No effects were observed in any other experimental condition.

Prenatal and perinatal fenarimol-induced genotoxicity in leukocytes of in vivo treated rats.

The identification of environmental compounds that have adverse effects on reproductive health and animal development is particularly challenging. Fenarimol, a systemic fungicide, is considered non or weakly genotoxic. However, its available toxicological data are controversial and incomplete. This study was conducted in rat in vivo to determine whether this compound (150 and 300 mg/kg) had adverse effects on DNA integrity in dams and pups after maternal subcutaneous exposure. The animals were exposed during early gestation (1-6 days), late gestation (last 6 days), or first 6 days of lactation. Findings on fenarimol genotoxicity showed an adverse effect when detected by the Comet assay, both in dams and pup, and state that animal sensitivity to fenarimol is higher during postnatal period. Since the DNA damage increases during the time of exposure (2 h to 6 days after the birth), our data on pups suggest that fenarimol can mainly act on cell DNA through direct exposure of litter via milk.

Toxicity and genotoxicity of surface water before and after various potabilization steps.

Many studies have revealed the presence of compounds with genotoxic activity in drinking water by means of short-term mutagenicity tests. In this study, the influence of the different steps of surface water treatment on the mutagenicity of drinking water was evaluated. Four different types of samples were collected: raw lake water, water after pre-disinfection with chlorine dioxide, water after filtration on granular activated carbon, and tap water. Water extracts underwent a bacterial toxicity test (Microtox test) and different in vitro genotoxicity tests: a test with Salmonella typhimurium strains, a Saccharomyces cerevisiae test, the SOS Chromotest with Escherichia coli and the Mutatox test with Vibrio fischeri. The Microtox test revealed high toxicity in the treated water samples. The disinfection steps increased the toxicity: the Mutatox test confirmed these results and the Salmonella/microsome test at the highest doses showed toxicity that could conceal mutagenicity. The SOS Chromotest was positive in all treated water samples without metabolic activation. In the test with S. cerevisiae both toxicity and genotoxicity generally increased during the water treatment steps, especially in cells without induction of cytochrome P450.

[Cytotoxicity and genotoxicity of drinking water of two networks supplied by surface water]. / Citotossicità e genotossicità di acque potabili di due impianti approvvigionati con acque superficiali.

OBJECTIVE: Evaluation of cytotoxic and genotoxic load of drinking water in relationship to the source of supplies, the disinfection process, and the piping system. SETTING: Two treatment/distribution networks of drinking water, the first (#1) located near the source, the second (#2) located near the mouth of a river supplying the plants. DESIGN: Water samples were collected before (F) and after (A) the disinfection process and in two points (R1 and R2) of the piping system. The samples, concentrated on C18, were tested for DNA damage in human leukocytes by the Comet assay and for gene conversion, reversion and mitochondrial mutability in Saccharomyces cerevisiae D7 strain. MAIN OUTCOME MEASURES: The approach used in this study is able to identify genotoxic compounds at low concentration and evaluate their antagonism/synergism in complex mixtures. RESULTS: Comet assay results show that the raw water quality depends on the sampling point, suggesting that a high input of environmental pollutants occurred during river flowing; they also show that the disinfection process can both detoxify or enhance biological activity of raw water according to its quality and that the piping systems do not affect tap water cytotoxic/genotoxic load. The yeast tests indicate the presence of some disinfection by-products effective on mitochondrial DNA. CONCLUSION: The biological assays used in this study are proven to be able to detect the presence of low concentrations of toxic/genotoxic compounds and assess the sources of their origin/production.

Cytotoxic and genotoxic effects of megazol, an anti-Chagas' disease drug, assessed by different short-term tests.

Cyto- and genotoxicity induced by drugs can limit the dose and duration of treatment, can adversely affect patient quality of life, and may be life-threatening. Two drugs are currently being used for treatment of the acute phase of Chagas' disease and both have serious undesirable effects. In this research, cyto- and genotoxic activity of the nitroimidazole-tiadiazole derivative CL 64855 2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole (megazol), a promising alternative drug, was evaluated in vitro with different short-term tests: (a) induction of recombination events and mutation in the yeast Saccharomyces cerevisiae D7 strain, with and without induction of cytochrome P-450; DNA damage (single and double strand breaks, alkali-labile sites, etc.) by the Comet assay in different mammalian cells. S. cerevisiae did not show a significant increase of mutant and recombinant event frequency, both with and without cytochrome P-450. On the other hand, the cytochrome complex appeared to detoxify the drug with respect to cytotoxicity. Results in rat and mouse fresh leukocytes showed a dose-response relation of drug-induced DNA damage. Findings in treated VERO cells suggested a complex treatment time-DNA damage relationship and the possible induction of repair mechanisms. Furthermore, bleomycin effects were increased in rat cells by simultaneous administration of megazol. Megazol shows different biological activity in relation to cellular types and experimental conditions (with or without cytochrome P-450, short/long time of exposure, with or without other genotoxins), thus suggesting a modulation of effectiveness by different physiological/biochemical conditions of cells. The findings could be useful to evaluate new megazol-derived compounds and to assess the risks/benefits relationship for each drug.

Bleomycin genotoxicity and amifostine (WR-2721) cell protection in normal leukocytes vs. K562 tumoral cells.

Recent advances in chemotherapy have focused on the benefit of high dose regimens, increasing the dose intensity of conventional chemotherapy. However, unacceptable cytotoxicity and genotoxicity on normal cells often impairs the proper management of patients. Phosphoaminothiol WR-1065, the active metabolite of amifostine, appears to protect normal cells and tissues against cytotoxic exposure to radiation or chemotherapeutic agents. Nevertheless, there is disagreement in findings on amifostine protection against bleomycin-induced severe side effects which have suggested that amifostine effectiveness against bleomycin-induced genotoxicity in normal leukocytes and tumour line cells K562 be studied. DNA damage was detected by single cell gel electrophoresis (or Comet) assay, a technique able to detect DNA strand breaks, alkali-labile sites and incomplete excision repair events in individual cells and which appears to be an ideal tool for assessing variability in response of different cell types in vitro. WR-2721 appears to selectively protect healthy leukocytes but not K562 tumoral cells. On the other hand, data on the inter- and intra-individual sensitivity to bleomycin and amifostine suggest that individual metabolic/genetic differences and other factors relating to lifestyle may be responsible for response variability. Application of the Comet assay in appropriate clinical settings to test the sensitivity of patients when undergoing chemotherapy appears possible.

Generalized anxiety disorder in referred children and adolescents.

OBJECTIVE: There are insufficient data on generalized anxiety disorder in children and adolescents. Symptoms and comorbidity of generalized anxiety disorder are described as a function of age, gender, and comorbidity in a consecutive series of referred children and adolescents. METHOD: One hundred fifty-seven outpatients (97 males and 60 females, 50 children and 107 adolescents, age range 7-18 years, mean age 13.4 +/- 2.7 years) were diagnosed as having generalized anxiety disorder, using historical information and a structured clinical interview (Diagnostic Interview for Children and Adolescents-Revised) according to the DSM-IV. RESULTS: Feelings of tension, apprehensive expectations, negative self-image, need for reassurance, irritability, and physical complaints were reported in more than 75% of the participants. Differences in symptomatology according to age and gender were nonsignificant. Depressive disorder was the most frequent comorbidity, being present in 56% of the patients. Comorbid anxiety disorders were present in about 75% of the patients, and 21% showed externalizing disorders. Subjects with comorbid depression had less anxiety comorbidity, subjects with comorbid separation anxiety disorder had higher rates of panic disorder, and subjects with comorbid externalizing disorders had higher rates of bipolar disorder. CONCLUSIONS: Referred children and adolescents with generalized anxiety disorder are heavily symptomatic and have frequent comorbidity. A more precise definition of the clinical picture may help early diagnosis and prevention of superimposed mental disorders.

Genetic polymorphism of drug-metabolizing enzymes and styrene-induced DNA damage.

A cross-sectional study was carried out on 48 workers exposed to styrene and 14 unexposed healthy controls in order to investigate the genotoxic potential of styrene exposure. DNA damage was assessed in peripheral blood leukocytes (WBCs) by the comet assay. Polymorphisms in glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) and the gene encoding microsomal epoxide hydrolase (EPHX) were characterized to assess their possible modifying role in styrene metabolism and subsequent DNA damage. Exposed workers showed significantly higher levels of DNA damage compared to controls. Among workers, the GSTM1 and GSTT1 polymorphisms significantly affected comet parameters. Subjects bearing a GSTM1pos genotype showed a significantly higher proportion of damaged nuclei compared to people lacking GSTM1-1 expression (GSTM1null), whereas GSTT1pos workers showed significantly lower DNA damage than GSTT1null individuals. Styrene-7,8-oxide (SO)-induced DNA damage was assessed in vitro in WBCs isolated from the healthy controls. A clear dose-response relationship at micromolar doses of SO was found for the whole group. WBCs collected from subjects bearing the homozygous wildtype GSTP1 genotype showed a significant protection compared to cells from subjects bearing at least one GSTP1 variant allele. The field survey confirms that styrene exposure is associated with increased DNA damage and indicates a modulating role for GSTM1 and GSTT1 genotypes. In vitro experiments suggest that the extent of SO-induced DNA strand breaks depends, at least in part, on interindividual differences in GSH-conjugation capabilities.