Biomedical subjects

M Fenech

Publications and source records attributed to M Fenech.

At least 19 recordsLinked to original sources

PubMed2006

Investigations into the relationship between hemodynamics and vascular alterations in an established arteriovenous fistula.

Arteriovenous fistula are specific vessels created by a vascular operation in order to provide sufficient blood access for extracorporeal circulation in hemodialysis. They are subject to numerous pathologies that may be caused by hemodynamic effects. To better understand these effects, a specific patient's arteriovenous fistula was reconstructed from computed tomography angiography. Computational fluid dynamics software made it possible to solve fluid mechanics equations under physiological conditions. An accurate map of unsteady velocity profiles and wall shear stress was drawn up. The computed velocity profiles were successfully confronted with Echo Doppler investigation. Selected regions with or without calcification, the end stage of wall alteration, were examined in terms of the mechanical constraints generated by blood flow. In contrast with other authors, we did not observe any association between calcification and areas of oscillating shear stress. Nevertheless, a statistical analysis of the whole vessel envelop and specific sites of calcification suggested a potential association between calcification and high temporal wall shear stress gradients.

Angiography↗

PubMed2005

Moderate acute intake of de-alcoholized red wine, but not alcohol, is protective against radiation-induced DNA damage ex vivo -- results of a comparative in vivo intervention study in younger men.

Moderate intake of wine is associated with reduced risk of cardiovascular disease and possibly cancer however it remains unclear whether the potential health benefits of wine intake are due to alcohol or the non-alcoholic fraction of wine. We therefore tested the hypothesis that the non-alcoholic fraction of wine protects against genome damage induced by oxidative stress in a crossover intervention study involving six young adult males aged 21-26 years. The participants adhered to a low plant phenolic compound diet for 48 h prior to consuming 300 mL of complete red wine, de-alcoholized red wine or ethanol on separate occasions 1 week apart. Blood samples were collected 0.5, 1.0 and 2.0 h after beverage consumption. Baseline and radiation-induced genome damage was measured using the cytokinesis-block micronucleus assay and total plasma catechin concentration was measured. Consumption of de-alcoholized red wine significantly decreased the gamma radiation-induced DNA damage at 1 and 2 h post-consumption by 20%. In contrast alcohol tended to increase radiation-induced genome damage and complete wine protected against radiation-induced genome damage relative to alcohol. The observed effects were only weakly correlated with the concentration of total plasma catechin (R=-0.23). These preliminary data suggest that only the non-alcoholic fraction of red wine protects DNA from oxidative damage but this effect cannot be explained solely by plasma catechin.

Adult↗

PubMed2005

Ovulation failure and double ovulation in dairy cattle: risk factors and effects.

Ovulation failure and double ovulation rates were examined in 1917 inseminations performed in high-yielding dairy cows under standard commercial conditions. The ovulation rate was determined 11 days post-insemination by ultrasound detection of at least one corpus luteum in the ovaries. Analyzing the double ovulation and pregnancy rates, the study population consisted only of ovulated cows (n = 1792). Data were analyzed using logistic regression methods. A failure to ovulate was recorded in 125/1917 (6.5%) services: 82/663 (12.4%) during the warm and 43/1254 (3.4%) during the cool period. Based on the odds ratios, the risk of ovulation failure was 3.9 times higher for inseminations performed during the warm period. No significant effects of estrous synchronization, milk production and days in milk at AI, and service and lactation number on ovulation failure were found. Double ovulation was recorded in 277/1792 (15.5%) services: 146 (52.7%) unilateral double ovulations (42.5% left versus 57.5% right); 115 (41.5%) bilateral double ovulations; and 16 (5.8%) triple ovulations. Double ovulation was recorded in 72 (12.4%) and 205 (16.9%) AI during the warm and the cool period, respectively. The percentages of double ovulation for first, second and third or more lactations were 6.7, 16.6 and 25%, respectively. Double ovulation rates for early (less than 90 days), mid- (90-150 days) and late (more than 150 days) lactation periods were 13, 20.7 and 14.2%, respectively. Reaching estrus during the warm period decreased the likelihood of double ovulation by a factor of 0.86; the risk of double ovulation was lower in cows with higher milk production (a 1 kg increase in milk yield led to a 0.97-fold reduced risk of double ovulation); cows in their second and in their third or more lactations showed a likelihood of double ovulation (using the first lactation as reference) increased by factors of 3.4 and 5.6, respectively; and reaching estrus during the early and late lactation period was related to a decreased probability of double ovulation (using the mid-lactation period as reference) by factors of 0.56 and 0.84, respectively. No significant effects of synchronization and service number on the double ovulation rate were found. Pregnancy was recorded in 914/1792 (51%) services: rates of 53.5% (811/1515) were recorded for single ovulations; 37.2% (103/277) for double ovulations: 28.8% (42/146) for unilateral double ovulations; 45.2% (52/115) for bilateral double ovulations; and 56.3% (9/16) for triple ovulations. The likelihood of pregnancy diminished in cows: inseminated during the warm period (by a factor of 0.5); inseminated by one particular bull (by a factor of 0.33); with higher milk production (a 1 kg increase in milk yield decreased the probability of pregnancy by a factor of 0.98); or undergoing unilateral (by a factor of 0.31) and bilateral (by a factor of 0.64) double ovulation. Logistic regression analysis indicated no significant effects of synchronization, days in milk, lactation number and service number on pregnancy rate. Collectively, our results indicate that cows showing estrus in conditions of heat stress had a high risk of ovulation failure. The effect of milk production on double ovulation was negative, whereas lactation number was positively correlated with this factor; the highest incidence of double ovulation occurring during the mid-lactation period.

Animals↗

PubMed2004

Comparison of DNA damage and repair following radiation challenge in buccal cells and lymphocytes using single-cell gel electrophoresis.

PURPOSE: To develop a reproducible single-cell gel electrophoresis assay for DNA damage and repair in buccal mucosa and sublingual exfoliated cells. MATERIALS AND METHODS: Buccal mucosa and sublingual cells and lymphocytes from six individuals (three males, three females, aged 34-45 years) were challenged with increasing doses of gamma-rays. DNA strand breaks and DNA repair were measured using the single-cell gel electrophoresis assay. RESULTS: Baseline DNA strand breaks were significantly greater in buccal mucosa and sublingual cells compared with lymphocytes. Buccal mucosa and sublingual cells did not differ from each other with respect to induction of DNA strand breaks by 2 or 4 Gy gamma-rays. However, they showed a smaller increase in gamma-ray-induced DNA strand breaks compared with lymphocytes (32-53% less than lymphocytes; ANOVA p<0.0001). Unlike lymphocytes, which repaired 83% of DNA strand breaks, buccal mucosa and sublingual cells exhibited only a minimal capacity for DNA repair (approximately 0-14% of the level in lymphocytes). CONCLUSIONS: Buccal mucosa and sublingual cells exhibit an apparent resistance to the expression of radiation-induced DNA strand breaks in vitro and an apparent lack of DNA strand break repair in the single-cell gel electrophoresis assay.

Adult↗

PubMed2003

Reproductive functional anatomy and oestrous cycle pattern of the female brush-tailed porcupine (Atherurus africanus, Gray 1842) from Gabon.

In the present study, we examined certain features of the functional anatomy of the female genital tract of the wild brush-tailed porcupine (Atherurus africanus) to obtain data on the reproductive biology of this African forest rodent. Two consecutive experiments were performed. The aim of the first was to establish macroscopic and microscopic features of the genital organs, and to explore correlations between predominant ovarian structures and vaginal contents in 20 wild, mature females. In the second experiment, we inspected the external genitalia and vaginal smears of a further 10 females in captivity on a daily basis for 90 days. The uterus of the brush-tailed porcupine is bicornuate and composed of two separated uterine horns, a uterine body and cervix. The genital tract does not present a vaginal vestibule. Thus, there is no portion common to genital and urinary tracts. Females in the follicular phase of the oestrous cycle showed increased cornification of the vaginal epithelium and a high density of eosinophilic cells in vaginal smears. The vulva and vaginal opening were open, reddish and tumefacted. In luteal phase or in pregnancy, epithelial cornification and eosinophilic features were notably reduced and the vagina presented a pale, non-tumefacted vulva and a vaginal closure membrane. Females in captivity showed spontaneous cycles, a polyoestrous reproduction pattern and, based on features of the external genitalia and vaginal smears, their oestrous cycle length was 27.1+/-6.4 days (n=12).

Animals↗

PubMed2003

HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures.

Criteria for scoring micronuclei and nucleoplasmic bridges in binucleated cells in the cytokinesis-block micronucleus assay for isolated human lymphocyte cultures are described in detail. Morphological characteristics of mononucleated cells, binucleated cells, and multinucleated cells as well as necrotic and apoptotic cells and nuclear buds are also described. These criteria are illustrated by a series of schematic diagrams as well as a comprehensive set of colour photographs that are of practical assistance during the scoring of slides. These scoring criteria, diagrams and photographs have been used in a HUman MicronNucleus (HUMN) project inter-laboratory slide-scoring exercise to evaluate the extent of variability that can be attributable to individual scorers and individual laboratories when measuring the frequency of micronuclei and nucleoplasmic bridges in binucleated cells as well as the nuclear division index. The results of the latter study are described in an accompanying paper. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories so that results from different laboratories for the cytokinesis-block micronucleus assay may be more comparable in the future.

Cell Division↗

PubMed2002

Risk factors for postpartum ovarian cysts and their spontaneous recovery or persistence in lactating dairy cows.

Cystic ovarian disease is a major cause of reproductive failure and economic loss for the dairy industry. Many cysts that develop during the early postpartum period regress spontaneously. However, it is difficult to decide at what point it would be more cost effective to treat ovarian cysts than to wait for spontaneous recovery. The objective of this study was to analyze risk factors for the development of the ovarian cystic condition during early and late postpartum, and for its persistence or recovery during the pre-service period in lactating dairy cows. Using multiple logistic regression, we analyzed data derived from 873 lactating dairy cows from a single herd. An ovarian cyst was diagnosed if it was possible to observe a single follicular structure with an antrum diameter > or = 25 mm in the absence of a corpus luteum in three sonograms performed at 7-day intervals. The cystic condition was denoted as early if the cyst was diagnosed 43-49 days postpartum, and late if detected 57-63-day postpartum. Spontaneous cyst regression before 60-day postpartum was regarded as early cystic recovery. For the early cystic group, there were no significant effects of lactation number, body condition score on prepartum Day 60, at parturition or on postpartum Day 30, or of body condition loss from parturition to 30-day postpartum. Cows calving in summer were 2.6 times more likely to develop ovarian cysts than those giving birth in winter. The risk of having a cyst was 1.9 times higher in cows with an abnormal puerperium. A 1-kg increase in milk yield raised the risk of cysts by a factor of 1.05. A 1-unit increase in body condition score (scale from 1 to 5) from prepartum Day 60 to parturition increased the risk of cyst development 8.4 times. Milk production and lactation number were negatively correlated with spontaneous early cyst recovery. A 1-kg decrease in milk production increased the probability of cyst recovery by a factor of 1.06, and a 1-unit drop in lactation number was associated with a 1.4-fold increased probability of cyst recovery. For the late cystic group, there were no significant effects of abnormal puerperium and body score data, except for a prepartum change in body score. Calving season (Odds ratio: 2.3), lactation number (Odds ratio: 1.36), increased milk production (Odds ratio: 1.05) and increased body condition score during the prepartum period (Odds ratio: 4.3) were all related to an increased risk of ovarian cysts. The probability of having a late cyst was 36.6 times greater in cows with early cysts. These findings suggest that it would be profitable to treat multiparous cows having cysts very early in the postpartum period, while treatment of primiparous cows should be delayed, at least until the end of the pre-service period, to provide the opportunity for spontaneous recovery.

Animals↗

PubMed2002

Micronutrients and genomic stability: a new paradigm for recommended dietary allowances (RDAs).

Diet as a key factor in determining genomic stability is more important than previously imagined because we now know that it impacts on all relevant pathways, namely exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis. Current recommended dietary allowances for vitamins and minerals are based largely on the prevention of diseases of deficiency such as scurvy in the case of vitamin C. Because diseases of development, degenerative disease and aging itself are partly caused by damage to DNA it seems logical that we should focus better our attention on defining optimal requirements of key minerals and vitamins for preventing damage to both nuclear and mitochondrial DNA. To date, our knowledge on optimal micronutrient levels for genomic stability is scanty and disorganised. However, there is already sufficient evidence to suggest that marginal deficiencies in folate, vitamin B12, niacin and zinc impact significantly on spontaneous chromosome damage rate. The recent data for folate and vitamin B12 in humans with respect to micronucleus formation in blood and epithelial cells provide compelling evidence of the important role of these micronutrients in maintenance of genome integrity and the need to revise current RDAs for these micronutrients based on minimisation of DNA damage. Appropriately designed in vitro studies and in vivo placebo controlled trials with dose responses using a complementary array of DNA damage biomarkers are required to define recommended dietary allowances for genomic stability. Furthermore these studies would have to be targeted to individuals with common genetic polymorphisms that alter the bioavailability of specific micronutrients and the affinity of specific key enzymes involved in DNA metabolism for their micronutrient co-factor. That there is a need for an international collaborative effort to establish RDAs for genomic stability is self-evident.

DNA Damage↗

PubMed2002

Reversibility of artifacts of fluid volume measurements by bioimpedance caused by position changes during dialysis.

The effect of temporary position changes, sitting up from supine, on extracellular (ECW) and intracellular (ICW) resistances and fluid volumes calculated from whole body bioimpedance using a Xitron 4200 impedancemeter was investigated on 8 patients during dialysis for a total of 11 tests. It was found that ECW resistance decreased instantaneously by an average of 2.3% when the patient sits up, due to plasma and interstitial fluid shift into the legs which decreases leg resistance, the major contributor to total resistance. This drop in resistance is incorrectly interpreted by the device as an increase in ECW volume which averages 235 ml. But this effect is completely reversible and both ECW resistance and fluid volume rapidly resume their normal course when the patient returns to his initial position. No significant variation in ICW resistance was observed in any of the patients at the position change. We conclude that segmental impedance, which has been proposed to minimize this artifact, is not advisable in dialysis monitoring and that it is simpler to ignore or switch off measurements during the position change so that later data are not affected by it.

Aged↗

PubMed2001

Recommended dietary allowances (RDAs) for genomic stability.

Diet as a key factor in determining genomic stability is more important than previously imagined because we now know it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis. Current recommended dietary allowances for vitamins and minerals are based largely on the prevention of diseases of deficiency such as scurvy in the case of Vitamin C. Because diseases of development, degenerative disease and ageing itself are partly caused by damage to DNA, it seems logical that we should focus better our attention on defining optimal requirements of key minerals and vitamins for preventing damage to both nuclear and mitochondrial DNA. To date our knowledge on optimal micronutrient levels for genomic stability is scanty and disorganised. Appropriately designed placebo, controlled trials are required to define recommended dietary allowances for genomic stability. Recently, it has been shown that above RDA intakes of folic acid and Vitamin B12 are required to reduce the micronucleus index in humans by 25%. In the future, clinical trials with a defined wider array of complementary DNA damage end-points would be necessary. That there is a need for an international collaborative group to establish RDAs for genomic stability is self-evident and this paper is a call for such a process to begin.

Ascorbic Acid↗

PubMed2001

Vitamins/minerals and genomic stability in humans.

Recommended dietary allowances (RDAs) of micronutrients have been traditionally derived as those levels necessary to prevent symptoms of deficiency diseases. There is increasing evidence that higher levels of many such micronutrients may be necessary for various DNA maintenance reactions, and that the current RDAs for some micronutrients may be inadequate to protect against genomic instability. Supplementation of a normal diet, with either vitamins and/or minerals or with isolated plant polyphenols, is becoming increasingly common in most Western populations. However, there is no clear agreement as to how much supplementation should occur, if at all, and genotypic differences are not accounted for. The 14 mini-reviews in this special issue summarise the role of specific micronutrients in various aspects of DNA maintenance: DNA synthesis, DNA repair, DNA methylation, gene mutation, chromosome breakage, chromosome segregation, gene expression, oxidative stress, necrosis and apoptosis. Evidence has been collated from mammalian and human experiments, both using in vitro cultures and in vivo approaches. Authors were asked to critically assess the strength of evidence as to whether the micronutrient can affect genomic stability in humans at realistic intake levels, and to estimate optimal dietary ranges where possible. Information on further research necessary is also documented. These reviews are an essential step towards a definition of RDAs designed to maintain genomic stability.

DNA↗

PubMed2001

The role of folic acid and Vitamin B12 in genomic stability of human cells.

Folic acid plays a critical role in the prevention of chromosome breakage and hypomethylation of DNA. This activity is compromised when Vitamin B12 (B12) concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation and DNA hypomethylation. In vivo studies show that Vitamin B12 deficiency and elevated plasma homocysteine are significantly correlated with increased micronucleus formation. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is >227nmol/l. Intervention studies in humans show: (a) that DNA hypomethylation, chromosome breaks, uracil misincorporation and micronucleus formation are minimised when red cell folate concentration is >700nmol/l folate; and (b) micronucleus formation is minimised when plasma concentration of Vitamin B12 is >300pmol/l and plasma homocysteine is <7.5micromol/l. These concentrations are achievable at intake levels in excess of current RDIs i.e. more than 200-400microgram folic acid per day and more than 2microgram Vitamin B12 per day. A placebo-controlled study with a dose-response suggests that based on the micronucleus index in lymphocytes, an RDI level of 700microgram/day for folic acid and 7microgram/day for Vitamin B12 would be appropriate for genomic stability in young adults. Dietary intakes above the current RDI may be particularly important in those with extreme defects in the absorption and metabolism of these Vitamins, for which ageing is a contributing factor.

Cells, Cultured↗

PubMed2001

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Animals↗

PubMed2001

Fluid volumes determination by impedance spectroscopy and hematocrit monitoring: application to pediatric hemodialysis.

A method for extracting fluid volumes from multifrequency bioimpedance, which takes into account the body geometry and the presence of nonconducting elements, was tested on 12 young dialyzed patients against correlations for total body water volumes (TBW) from Watson et al. and Humes et al. Our calculations of TBW from impedance were found to overestimate Humes' values by 0.25 L (0.8%) postdialysis and by 2.08 L (6.5%) predialysis. Extracellular water (ECW) was found to contribute an average of 93% of ultrafiltered volume. Intracellular water volume (ICW) determination from impedance was found to be too imprecise to predict its variation during dialysis; therefore, ICW variations were calculated as the difference between ultrafiltration and ECW changes. The continuous recording of hematocrit by an optical device monitored changes in plasma and interstitial volumes. In most cases, ultrafiltration was compensated mainly by a contribution from interstitial fluid, and the drop in plasma volume was generally moderate.

Adolescent↗

PubMed2001

Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes.

Human biomonitoring of early genetic effects requires accurate, sensitive and, if possible, easy and not too time-consuming methodologies to assess mutations. One of the most promising methodologies at the present time is the cytokinesis block micronucleus (MN) assay (CBMN), which detects both chromosome breakage and chromosome loss in once-divided binucleated (BN) cells. Many studies have been published with this methodology, but before its extensive application is recommended, it is necessary to evaluate its strengths and limitations. Recently, Fenech et al. reviewed the advantages of the CBMN assay for biomonitoring purposes. However, up to now information present in mononucleated (MONO) cells has rarely been taken into account, although it might be complementary to that assessed in BN cells. Indeed, MONO cells should indicate damage which was present in vivo before the start of culture and BN cells may contain pre-existing micronuclei (MNi) plus lesions which are expressed as MNi during in vitro culture. To address this question, the objectives of this paper were as follows. (i) To situate the CBMN assay in a historical and mechanistic perspective. (ii) To consider whether impaired mitotic capacity in vitro may be responsible for false negative biomonitoring studies if MN in MONO cells are not taken into account in the CBMN test. The following factors were considered: division delay for repair and mitotic block, in vitro apoptosis and necrosis of damaged cells, mitotic slippage and correlation between MN expression in vitro versus in vivo. (iii) To analyse the factors which may cause a negative result in the CBMN assay in biomonitoring when exposure to specific genotoxins is evident. The specific effects of aneugens and of adaptive responses to chronic low level exposure were examined. (iv) To compare the sensitivity of MONO and BN cells in relation to the genotoxic mechanism. (v) To propose an adequate sampling scheme to study MN in both MONO and BN cells. It was concluded that a more comprehensive assessment of DNA damage may be achieved if the CBMN assay includes measures of: (i) MNi in MONO cells; (ii) MNi in BN cells; (iii) apoptotic cells; (iv) necrotic cells. It is probable that the 24 h post-phytohaemagglutinin time point may be the optimal time to assess the frequency of MNi in MONO cells, apoptotic cells and necrotic cells. It is also practical to include these measures when scoring MNi in BN cells after cytokinesis block.

Apoptosis↗

PubMed2001

Preliminary study of the genotoxic potential of homocysteine in human lymphocytes in vitro.

Homocysteine (Hcy), an immediate precursor of methionine (Met), is considered a risk factor for cardiovascular disease, Alzheimer's disease and neural tube defects. Hcy concentration is also reported to correlate positively with the micronucleus index in lymphocytes in vivo, a marker of chromosome damage. However, it is unclear whether Hcy is genotoxic or simply a biomarker of folate deficiency, a known cause of chromosome damage. We investigated whether high concentrations of Hcy are genotoxic to human lymphocytes in vitro using the cytokinesis-block micronucleus assay. Eighteen lymphocyte cultures were initiated in Met-free and serum-free RPMI 1640 medium for each of four male volunteers aged 22-23 years. At 0, 24, 44 and 72 h, cultures were spiked with L-Hcy or L-Met to achieve concentrations ranging between 50 and 400 microM. The concentration of Hcy at 96 h ranged from 19.45 +/- 2.34 to 149.02 +/- 28.16 microM in Hcy cultures and 0.91 +/- 0.17 to 2.15 +/- 0.9 microM in Met cultures spiked with 50 and 400 microM of metabolite, respectively. Forty-four hours after mitogen stimulation, cytokinesis was inhibited with cytochalasin B. After 96 h, cells were transferred to microscope slides and the frequency of micronucleated-binucleate and necrotic cells was scored. Neither Hcy (P = 0.24) nor Met (P = 0.93) had an apparent dose effect on micronucleus frequency. However, when data were pooled, micronucleus frequency was moderately higher (50.1%) in Hcy- than in Met-spiked cultures (P = 0.04; paired t-test). Hcy concentration was positively correlated with necrosis (P < 0.0005; r(2)= 0.276), however, when data were pooled, levels of necrosis were higher in Met- than in Hcy-spiked cultures (P= 0.01; paired t-test). Further research is required to define more clearly the genotoxic and cytotoxic potential of homocysteine and its metabolites.

Adult↗

PubMed2001

Normal human lymphocytes exhibit a wide range of methionine-dependency which is related to altered cell division but not micronucleus frequency.

The underlying cause(s) of methionine-dependency and its relevance to cancer remains unclear. We aimed to determine whether (i) normal human lymphocytes exhibit methionine-dependency, (ii) baseline levels of genetic damage are related to methionine-dependency and (iii) methionine-dependence can be explained, in part, by common polymorphisms in methionine synthase and methylenetetrahydrofolate reductase (MTHFR). Genetic damage was measured in lymphocytes of 52 volunteers (29--65 years) using the cytokinesis-block micronucleus assay. Methionine-dependency was assessed by culturing cells in serum-free media containing 0.1 mM L-methionine and 0 mM D,L-homocysteine (met(+)hcy(-)) or 0 mM L-methionine and 0.2 or 0.4 mM D,L-homocysteine (0.2/0.4-hcy(+))(met(-)hcy(+)). Mitogenesis was stimulated with phytohaemagglutinin. Cytokinesis was inhibited by adding cytochalasin B at 44 h. Ninety-six hours after PHA, cells were transferred to microscope slides. Cell proliferation was measured by counting binucleated cell frequency and calculating nuclear division index. Volunteers were classified into tertiles of methionine-dependence according to the growth of their cells in met(-)hcy(+) media (relative to growth in met(+)hcy(-) media). Average cell division, as a percentage of division in met(+)hcy(-) media, was approximately 5, 26 and 70% in 0.2-hcy(+) media and 29, 70 and 142% in 0.4-hcy(+) media for the high, mid and low tertiles of methionine-dependence, respectively. Micronucleus frequency did not vary between these tertiles (P > 0.6). In both met(+)hcy(-) and met(-)hcy(+) media, cell division was not affected by polymorphisms in MTHFR (C677T, A1298C) or methionine synthase (A2756G). Cell division in met(-)hcy(+) media was negatively correlated with division in met(+)hcy(-) media (P = 0.05 and 0.007 for 0.2 and 0.4-hcy(+), respectively). Methionine-dependent lymphocytes had higher levels of cell proliferation in met(+)hcy(-) media than methionine-independent lymphocytes (P = 0.089 and 0.01 for 0.2 and 0.4-hcy(+), respectively). However, this difference was not apparent in previous experiments when cells were grown in media containing 10% fetal calf serum. These findings show that there is a wide inter-individual variation in the degree of methionine-dependency of normal human lymphocytes in vitro. Methionine-dependency does not appear to alter the risk for chromosomal mutation as measured by the micronucleus assay. We discuss the possible relevance to cancer of increased cell division in methionine-dependent cells under methionine-replete and serum-free media conditions.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2001

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.

Adolescent↗