Roberto Barale

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‰ and the interquartile range was between 3 and 12‰. 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. Environ. Mol. Mutagen. 37:31–45, 2001

Association of ABCB1/MDR1 and OPRM1 Gene Polymorphisms With Morphine Pain Relief

The pharmacokinetics and pharmacodynamics of morphine are under the control of several polymorphic genes, which can account for part of the observed interindividual variation in pain relief. We focused on two such genes: ABCB1/MDR1, a major determinant of morphine bioavailability, and OPRM1, which encodes for the μ‐opioid receptor, the primary site of action for morphine. One hundred and forty‐five patients of Italian origin undergoing morphine therapy were genotyped for the single‐nucleotide polymorphism (SNP) C3435T of ABCB1/MDR1 and for the A80G SNP of OPRM1. Pain relief variability was significantly (P<0.0001) associated with both polymorphisms. Combining the extreme genotypes of both genes, the association between patient polymorphism and pain relief improved (P<0.00001), allowing the detection of three groups: strong responders, responders, and non‐responders, with sensitivity close to 100% and specificity more than 70%. This study provides a good example of the possible clinical use of pharmacogenetics.

Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects

Microscopic examination of individual human lymphocytes embedded in agarose, subjected to electrophoresis and stained with a fluorescent DNA-binding dye, provides a novel way of measuring DNA damage as extent of migration of DNA fragments, mainly single-strand breaks. With this relatively simple method, DNA damage arising as a consequence of smoking, age and other factors was examined in peripheral human lymphocytes from 100 healthy individuals living in Pisa (Italy). The extent of DNA migration was found to be significantly increased by smoking. It is noteworthy that the effect of smoking was more significant in men than in women and that DNA migration was similar in the young and in the older people. SCE analysis did not reveal any significant effect of smoking, sex or age in the same population, suggesting a higher responsiveness of the comet test to DNA-damaging agents.

Herbicide-induced DNA damage in human lymphocytes evaluated by the single-cell gel electrophoresis (SCGE) assay.

The genotoxicity of the herbicides, alachlor, atrazine, maleic hydrazide, paraquat and trifluralin has been evaluated in the single-cell gel electrophoresis (SCGE) assay by using human peripheral blood lymphocytes. All treatments were conducted with and without the presence of an external bioactivation source (S9 mix). The results indicate that all the herbicides tested are able to give positive results by increasing the comet tail length, which would confirm both the genotoxicity of the herbicides and the sensitivity of the assay in front of these chemicals. Alachlor and atrazine give similar results in treatments with and without S9, while when the S9 mix was not used paraquat and trifluralin genotoxicity was higher. On the other hand, although maleic hydrazide genotoxicity was higher when S9 mix was used at normal pH (7.4), our data show that its genotoxicity depends largely on the pH solution, increasing as the pH decreased.

Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1,650 subjects in an Italian population: II. Contribution of sex, age, and lifestyle.

Sister chromatid exchange (SCE) and micronuclei (MN) analysis was carried out on 1,650 healthy individuals living in Pisa and in two nearby small cities, Cascina and Navacchio (Ca‐Na). The effect of smoking on SCEs was linearly correlated with the number of cigarettes per day, and an increase of 7.3% SCEs was detectable for as few cigarettes as 1–10/day. Ex‐smokers showed intermediate mean values of SCEs (8.09 ± 1.88) in comparison with never smokers (7.54 ± 1.61) and current smokers (8.45 ± 1.94). Mean values of SCEs of ex‐smokers decreased linearly with time of smoking cessation, reaching the mean values of never smokers within 8 years. The extent of SCE decrease was inversely proportional to the number of cigarettes previously smoked. No interaction between smoking habits and coffee or alcohol drinking on SCEs was observed. A borderline (P = 0.053) increase in mean SCE values in coffee drinkers (more than 3 cups/day) was found. The age effect on SCEs was remarkable in Ca‐Na, but not in Pisa donors. Job type was not associated with significant modification of mean values of SCEs. Multiple logistic regression analysis revealed a statistically significant association between the proportion of high frequency cells (HCF) outliers and coffee consumption. Age and sex appeared to be by far the most important variables associated with modifications in MN frequency, which increased by 0.04 and 0.02 per year in males and females, respectively. Children and young donors (age ≤ 40 years) showed lower MN frequency regardless of sex, whereas sex appeared to determine a significantly higher increase of MN only in females older than 40 years. In contrast, in males the MN rate by age tended to level off after the age of 30–50. MN frequencies of Pisa blue‐ and white‐collar workers were statistically significantly higher than in students (+0.71 and +0.55, respectively). Smoking did not determine any increase of MN frequency. A total lack of correlation (P = 0.913) between MN and SCEs was observed. Environ. Mol. Mutagen. 31:228–242, 1998

Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects

The comet test (single cell gel electrophoresis, SCGE) appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. Previously, we analyzed the degree of DNA damage in peripheral human lymphocytes from 100 healthy subjects living in Pisa (Italy) taking into account age, gender and smoking habit, and we also reported some results aiming at the assessment of the comet test (Betti el al., 1994). In addition, SCE analysis was carried out in order to compare the two endpoints. Because of the interesting results obtained, the present study was extended to 200 individuals, and data analyzed included information concerning number of cigarettes smoked a day, tar/cigarette and job. Data obtained confirmed that the SCGE is more sensitive than SCE in revealing smoking habit effects but comet induction did not seem to be related to the amount of cigarette tar inhaled. Moreover, sampling time was found to play a greater role in the comet assay as compared to SCE. Job position did not significantly influence SCE mean/subject or comet length mean/subject.

Cancer prevention in Europe: The Mediterranean diet as a protective choice

In the coming years, European death rates because of cancer will further decline, but the overall number of cases will increase, mostly as a consequence of the ageing of the population. The target for cancer prevention in Europe will remain a healthy diet and control of obesity in addition to a decrease in smoking. A healthy diet model in European countries is the traditional Mediterranean diet, which is based on abundant and variable plant foods, high consumption of cereals, olive oil as the main (added) fat, low intake of (red) meat and moderate consumption of wine. The Mediterranean diet is associated with a reduced risk of cardiovascular disease and cancer. The biological mechanisms for cancer prevention associated with the Mediterranean diet have been related to the favourable effect of a balanced ratio of omega 6 and omega 3 essential fatty acids and high amounts of fibre, antioxidants and polyphenols found in fruit, vegetables, olive oil and wine. The Mediterranean diet also involves a ‘Mediterranean way of drinking’, that is, regular, moderate consumption of wine mainly with food. This pattern of drinking increases longevity, reduces the risk of cardiovascular disease and does not appreciably influence the overall risk of cancer. However, heavy alcohol drinking is associated with digestive, upper respiratory tract, liver and breast cancers; therefore, avoidance or restriction of alcohol consumption to two drinks/day in men and one drink/day in women is a global public health priority.

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. environmental protection agency gene-tox program☆

The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.

Induction of micronuclei in gill tissue of Mytilus galloprovincialis exposed to polluted marine waters

The micronucleus assay in gill tissue of the mussel Mytilus galloprovincialis has been developed in our Laboratory to assess the mutagenic activity of compounds present in marine environments. The sensitivity of the test was assessed by performing mutagenic treatment for 48 h with the two standard compounds vincristine (VCR) (0.005, 0.01, 0.02 mg l−1), and benzo(a)pyrene (BaP) (0.025, 0.075, 0.225, 0.675 mg l−1). Since both tested chemicals produced significant increases in the number of micronucleated cells, animals were directly exposed to marine waters: mussels grown in clean water (control sample) were transferred to polluted areas and then collected weekly. Micronuclei frequencies of sampled mussels were significantly higher than the value of the control group.

Mutagenicity of industrial compounds styrene and its possible metabolite styrene oxide

Styrene and its presumed metabolite, styrene oxide, were tested for their mutagenic effect on a forward mutation system of yeast and of Chinese hamster cells, and on a gene-conversion system of yeast. Experiments with liver microsomal preparations and host-mediated assay with yeast were also carried out. Styrene oxide was mutagenic in all test systems. Styrene was mutagenic only in the host-mediated assay.