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M Fenech

Publications and source records attributed to M Fenech.

62 records · Page 4Linked to original sources
PubMed1987

Ageing in vivo does not influence micronucleus induction in human lymphocytes by X-irradiation.

To test the hypothesis that the age-related decline in the stability of the genome is the consequence of an increasing deficiency in DNA repair we compared the extent of chromosome damage, after X-irradiation, in lymphocytes from healthy young and old individuals, using the expression of micronuclei as the end-point. Micronuclei have been shown to increase in number with age and they were enumerated using a recently described and improved technique which involves measurement of micronuclei in cells that were blocked from performing cytokinesis. The level of X-ray-induced micronuclei in cytokinesis-blocked cells, after exposure to 75 cGy and 150 cGy, was measured by subtracting the base-line micronucleus frequency in the control unirradiated cultures from the observed micronucleus frequency in the irradiated cultures. There was no difference between the results for the young and old subjects thus indicating that cells from the aged subjects do not exhibit increased chromosomal instability following X-irradiation. These results suggest that repair of those DNA lesions that lead to chromosome breakage does not decline with age.

Adult↗
PubMed1986

Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation.

The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (less than 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.

Adult↗
PubMed1985

Solutions to the kinetic problem in the micronucleus assay.

The standard micronucleus assay as applied to lymphocyte cultures does not discriminate between non-dividing cells and cells that have divided. Micronuclei can only be expressed after nuclear division and because the proportion of dividing cells may vary from one culture to another the standard micronucleus assay can be very imprecise. To overcome this problem we have developed four new methods for scoring micronuclei only in lymphocytes that have divided once only. In the stathmokinetic method the proportion of dividing cells was measured by blocking them at metaphase with colchicine and the number of micronuclei was determined in a parallel culture. The ratio of micronucleus index and metaphase index gave the number of micronuclei per metaphase. With the flow cytometric method, the cells in S phase were allowed to incorporate bromodeoxyuridine and then allowed to divide. By virtue of their reduced fluorescence after staining with Hoechst 33342 it was possible to sort these cells out with a fluorescence activated cell sorter. The autoradiographic method employed incorporation of tritiated thymidine in cells in S phase so that after division these cells could be recognised by their labelled nuclei. In the cytokinesis-block method, dividing cells were inhibited from performing cytokinesis by exposing them to 3.0 micrograms/ml cytochalasin-B. Dividing cells were easily recognised by their binucleate appearance. Results from the four methods for micronuclei induced by X-rays were in close agreement, but the simplest and most precise of the methods was the cytokinesis-block method.

Adult↗
PubMedDate not supplied

The effect of donor age on spontaneous and induced micronuclei.

The spontaneous micronucleus yield in lymphocyte cultures from healthy donors aged 0-82 years was estimated at 72 h and 96 h of culture. At both 72 and 96 h there was a positive correlation of micronucleus expression with increasing age (p less than 0.001), with an approximately 4-fold increase in micronuclei in cultures from 80-year-old donors when compared to cultures from newborn donors. Since there is some evidence that the effect of DNA-damaging agents may increase with age, lymphocytes from individuals of various ages were exposed to X-rays and mitomycin C and micronuclei were scored after 72 and 96 h of culture. Micronucleus formation after exposure to these agents was, however, decreased in cells from elderly individuals, most likely due to kinetic differences between the lymphocytes of old and young individuals.

Adolescent↗
PubMedDate not supplied

Measurement of micronuclei in lymphocytes.

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.

Cell Division↗
PubMedDate not supplied

Sex is an important variable affecting spontaneous micronucleus frequency in cytokinesis-blocked lymphocytes.

The micronucleus frequency in cytokinesis-blocked lymphocytes of 152 females and 113 males aged between 20 and 89 years (minimum of 15 subjects per sex per decade) was compared. Marked differences in the micronucleus frequency of males and females were observed: (a) there was a greater dispersion in the results for females when compared to males in all age groups older than 40 years; (b) there was a significant positive correlation between micronucleus frequency and age in both sexes (p < 0.0001) but the slope of the linear regression line was steeper in females (slope = 0.499 micronuclei/year) compared to males (slope = 0.289 micronuclei/year) (p < 0.0045); (c) the micronucleus frequency in females (Mf) was significantly higher than the micronucleus frequency in males (Mm) in all decades examined (p < 0.05), the Mf,Mm ratio varied between 1.47 and 1.65 (mean +/- 1 SEM = 1.53 +/- 0.03) and showed no trend with age. These results suggest that an added mechanism, possibly the loss of X chromosomes, is contributing to the micronucleus frequency in females and highlights the importance of sex as a variable that has to be taken into consideration when interpreting data from cross-sectional studies utilising the cytokinesis-block micronucleus assay as a biomarker of chromosome damage.

Adult↗