Evaluation of the reticulocyte micronucleus assay in patients treated with radioiodine for thyroid cancer

In the case of accidental radiation exposure, biological dosimetry has an important role. Previous studies have indicated that the flow cytometric micronucleus (MN) assay in human transferrin receptor positive reticulocytes (Tf-Ret) in blood could be a sensitive biomarker for chromosome damage. In the present investigation, the utility and sensitivity of this method was studied in 44 young patients from Belarus, who were treated with 131I for thyroid cancer. Red marrow (RM) is the critical organ in radioiodine therapy (RIT). In our patients, it was exposed to 100-700 mSv low-dose rate irradiation within 2-4 days. About 3 days after 131I administration, the frequency of micronucleated-Tf-Ret (f(MN-Tf-Ret)) increases within 1 day to a maximum and declines in the following 2-5 days to its value before treatment. A total dose of 100 mSv was easily detectable. The sensitivity of the assay after acute irradiation may be 50 mSv. The method should be useful for monitoring individuals after a radiation accident, provided blood samples can be obtained within a few days after exposure. The time-course of f(MN-Tf-Ret) is interpreted using a model, which considers the exponential exposure of red marrow in RIT as well as the kinetics of erythroblast maturation and reticulocyte migration into the peripheral circulation. Similar modelling was done on published data of MN in immature mouse erythrocytes. Striking similarities in the kinetic and in the yield of MN-induction were found between these two species. This lends support for the use of the mouse as a model for the MN-induction in humans.     

Increased frequency of specific alleles of the c-Ha-ras gene in Japanese cancer patients

Summary We have examined the c-Ha-ras locus in 145 cancer patients of a mixed group and 164 normal individuals in Japan for restriction fragment length polymorphisms and compared the allele distributions in normal and cancer populations. The c-Ha-ras gene is highly polymorphic in Japanese as previously reported in Caucasians. Two rare alleles were found to be present with increased frequencies in Japanese cancer patients. These results suggest that genotype analysis of the c-Ha-ras gene could be used to detect cancer-prone individuals.     

Assessment of micronucleus frequency in normal oral mucosa of patients exposed to carcinogens

OBJECTIVE: To assess the presence of micronuclei in exfoliated oral mucosal cells collected from 3 anatomic sites in patients exposed to tobacco and alcohol. STUDY DESIGN: Smears were prepared with normal oral mucosal cells obtained from the lower lip, tongue border and floor of the mouth of 21 controls, 28 tobacco users and 19 tobacco/alcohol users. Slides were stained with Feulgen stain for quantification of micronucleated cells, karyorrhexis and "broken eggs." RESULTS: The groups were similar in terms of the mean number of micronucleated cells and cells undergoing karyorrhexis. In the comparison of anatomic sites, the mean number of cells undergoing karyorrhexis was higher on the lower lip than on the tongue border or floor of the mouth (all groups). A significantly higher number of broken eggs was observed in the control group when compared to the tobacco and tobacco/alcohol groups at all anatomic sites. CONCLUSION: The higher number of broken eggs in patients not exposed to tobacco and/or alcohol suggests that this nuclear alteration may be associated with DNA repair or a healthy mucosa. A trend toward an increased number of micronucleated cells was observed for tobacco and/or alcohol users at all anatomic sites.     

Investigation of insulin resistance gene polymorphisms in patients with differentiated thyroid cancer

We aimed to investigate insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), insulin-like growth factor binding protein-3 (IGFBP-3) genotypes, which are thought to be involved in the pathogenesis of many solid tumors and have thus far not been studied in patients with differentiated thyroid cancer (DTC). The study consisted of 93 patients diagnosed with DTC (79 females, 14 males) and 111 healthy control subjects (63 females, 48 males). The anthropometric measurements, lipid profiles, thyroid function tests and homeostatic model assessment (HOMA) as an indicator of insulin resistance (IR) of all patients were recorded. In addition IRS-1, IRS-2 and IGFBP-3 gene polymorphisms were determined by using polymerase chain reaction and restriction fragment length polymorphism. Hardy–Weinberg equilibrium was tested for each gene polymorphisms, and genetic effects were evaluated by the Chi Square test and multiple logistic regression. Homeostasis model assessment of insulin resistance (HOMA-IR), body mass index, waist circumference and serum total cholesterol levels were significantly higher in patients with DTC than in the control group. There was no difference between the two groups with respect to IRS-1, IRS-2 and IGFBP-3 gene polymorphisms. In addition, these gene polymorphisms were found to have no effect on lymph node metastases or tumor staging. While, obesity and increased HOMA-IR may be risk factors in DTC development, we suggest that IRS-1, IRS-2 and IGFBP-3 gene polymorphisms do not play an important role in pathogenesis of DTC.     

Effect of thiols on micronucleus frequency in gamma-irradiated mammalian cells

The effects of the thiols cysteamine, WR-1065, and WR-255591 on radiation-induced micronucleus (MN) frequency and cell killing were compared in cultured Chinese hamster ovary cells. MN were measured using the cytochalasin B assay of Fenech and Morley (1985), which minimizes the effect of cytokinetic perturbations on MN expression. The dose-response curves for MN induction were curvilinear both for control cells at doses between 1.5 and 4.5 Gy and for thiol-treated cells at doses between 3 and 9 Gy. Protection against MN induction by each thiol was independent of radiation dose. Furthermore, there was a close correlation between the degree of modification of MN induction and cell survival by each thiol, i.e., the MN frequency closely predicted the survival level regardless of the presence of absence of the thiols. A similar predictive relationship has also been reported by us for cell survival and DNA double-strand break (DSB) induction in this cell line following treatment with these same thiols. Collectively, these data support the hypothesis that, for DNA-repair-proficient mammalian cells treated with radiomodifying agents that do not alter DNA-repair processes, MN and DSB induction are predictive of the level of radiation lethality and of each other.     

Environmental exposure to benzene, micronucleus formation and polymorphisms in DNA-repair genes: a pilot study

This report is part of a biomarker study conducted in an Italian population with exposure to environmental benzene ranging from 1.43 to 31.41 μg/m3 (values from personal sampling). DNA damage induced by benzene is the crucial mechanism of its genotoxicity, which leads to chronic benzene poisoning, haematotoxicity and leukaemia. Therefore, genetic variation in DNA-repair genes may modulate susceptibility to benzene-induced DNA damage. In light of this, the effects of polymorphisms in DNA-repair genes (APEX1, hOGG1, NBS1, XPD, XRCC1, and XRCC3) on micronucleus (MN) formation as a biomarker of early biological effects were evaluated. A significantly higher median MN frequency was recorded in traffic wardens than in controls. However, none of the analysed polymorphisms was significantly associated with the median MN frequency. A gene-gender interaction was observed for the APEX1 genotype. The APEX1 variant genotype was associated with significantly lower median MN frequency in men, not in women. Statistical analysis did not reveal any association between the score of the protective alleles - hypothetically pushing the pathway towards optimal DNA-damage repair - and MN. Even though there are some limitations in the study, our results indicate that the general population may be exposed to benzene concentrations higher than the threshold level for air-quality standards in the European Union of 10 μg/m3. Furthermore, urban traffic wardens are exposed to significantly higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting that benzene might be implicated both as an environmental and occupational risk factor in leukaemia and other haematological diseases. In conclusion, this study suggest the need for (i) regular monitoring of traffic wardens for possible exposure to benzene, as a precautionary step to reduce the associated health risks, and (ii) more comprehensive studies in order to better elucidate the involvement of APEX1 genotypes in benzene genotoxicity.     

Influence of initial large dose on subsequent uptake of therapeutic radioiodine in thyroid cancer patients

Fifty-two patients with differentiated thyroid cancer, following thyroidectomy were studied by administering a quantity of up to 5 mCi of [¹³¹I]sodium iodide. In most of these patients, radioiodine uptake values obtained with the subsequent therapeutic dose were markedly lower than those observed with the initial doses. This observation was verified in seven of the patients with differentiated thyroid cancer, by measuring the radioiodine uptake with a second dose of 4.5 mCi of [¹³¹I]sodium iodide. Calculations showed that the major etiology was probably therapeutic irradiation of the thyroid by the first dose.     

Influence of the endogene TSH stimulation of thyroid volume increase in the patients after total thyroidectomy due to differentiated thyroid cancer

INTRODUCTION: The treatment-of-choice for differentiated thyroid carcinoma (DTC) is a total thyroidectomy with subsequent radioiodine therapy. In order to increase an iodine uptake in thyroid tissue remnants, the L-thyroxine withdrawal is required. It is recommended to achieve TSH levels higher than 25 mU/ml. As TSH is a known key factor in thyroid cell proliferation regulation, prolonged stimulation of the cells during L-thyroxine withdrawal can be a causative factor for a re-growth. Our aim was to assess the degree of thyroid re-growth in the patients after total thyroidectomy due to DTC and its possible clinical implications. MATERIAL AND METHODS: 23 patients operated due to papillary and follicular thyroid cancer were included into the study. Biochemical determinations and ultrasound thyroid imaging were performed (TSH, Tg) during suppressive L-thyroxine therapy as well as 4-5 weeks after the withdrawal. RESULTS: The mean volume of thyroid tissue remnants increased after withdrawal for substantial 30.1%. The difference was extremely significant. CONCLUSIONS: L-Thyroxine withdrawal in the patients after total thyroidectomy due to DTC can cause re-growth of the tissue remnants. The phenomenon may be of a clinical significance in the selected cases influencing therapeutic decisions.     

Increased micronucleus frequency in exfoliated cells of the buccal mucosa in hairdressers

Hairdressers are exposed daily to chemical substances, such as dyes, chemical straighteners and curling chemicals, which can be absorbed, inhaled or possibly ingested. We analyzed the frequency of micronuclei (MNC) in exfoliated cells of the buccal mucosa of 50 hairdressers and 50 controls in Pelotas, RS, Brazil. An assessment was carried out on the incidence of MNC, binucleated cells (BNC), broken egg cells (BEC), budding cells (BC), and the sum of anomalies (SA), in 2000 cells per individual. The data were analyzed with SPSS, using the Mann-Whitney U-test, α = 0.05. The mean number of anomalies in hairdressers was 2.02 ± 3.60 MNC; 8.50 ± 5.07 BNC; 9.06 ± 3.83 BEC; 0.32 ± 0.62 BC, and 19.90 ± 9.61 SA; in controls it was 0.36 ± 1.06 MNC; 5.20 ± 4.73 BNC; 5.92 ± 2.67 BEC; 0.10 ± 0.36 BC, and 11.58 ± 6.67 SA; the differences for all parameters were significant. The non-occupational factors did not significantly influence the alterations. A significant increase of BEC (P = 0.003) was observed in the hairdressers and SA (P = 0.033) in females. The lowest income level influenced MNC (P = 0.044), and the habit of not smoking influenced SA (P = 0.020). We concluded that exposure to substances used by hairdressers is genotoxic for men.     

Effect of nitric oxide on gamma-ray-induced micronucleus frequency in RAW264.7 cells

The effect of low-dose nitric oxide (NO) on gamma-ray-induced micronucleus (MN) frequency was investigated in RAW264.7 cells. Treatment of RAW264.7 cells with 0.25 mM sodium nitroprusside (SNP), a chemical NO donor, reduced the frequency of micronuclei induced by 5 Gy gamma rays by 43 to 45% between 3 and 12 h post-treatment. This effect was blocked by carboxy-PTIO, suggesting that NO may play a role in the reduction of radiation-induced MN frequency. To examine possible mechanisms underlying this effect, we first looked at changes in the antioxidant system after SNP treatment. A significant increase in intracellular glutathione (GSH) was seen in SNP-treated cells between 3 and 12 h post-treatment. Depletion of GSH with buthionine sulfoximine (BSO) increased the gamma-ray-induced increase in MN frequency. Detailed studies using various inducers of intracellular GSH suggested that GSH induction has a partial role in the reducing effect of NO on the gamma-ray-induced MN frequency. Next, the effect of NO on DNA repair and replication systems was examined. Wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK), dose-dependently inhibited the reducing effect of NO, while caffeine, an inhibitor of ATM kinase and ATR kinase, did not. DNA-PK activity was increased by NO treatment. Etoposide, a topoisomerase II inhibitor, dose-dependently blocked the effect of NO in reducing the gamma-ray-induced MN frequency. These results suggest that the mechanisms of the effect of NO on the gamma-ray-induced MN frequency include elevation of GSH and up-regulation of DNA-PK activity for repairing double-strand breaks. NO may act as a signal for repair systems, e.g. for nonhomologous recombination and for the replication system in S phase, to reduce the MN frequency.     

Influence of variants in the NPY gene on obesity and metabolic syndrome features in Spanish children

Variants in the neuropeptide Y (NPY) gene have been associated with obesity and its traits. The objective of the present study was to evaluate the association of single nucleotide polymorphisms (SNPs) in the NPY gene with obesity, metabolic syndrome features, and inflammatory and cardiovascular disease (CVD) risk biomarkers in Spanish children. We recruited 292 obese children and 242 normal-body mass index (BMI) children. Height, weight, BMI, waist circumference, clinical and metabolic markers, adipokines, and inflammatory (PCR, IL-6, IL-8 and TNF-α) and CVD risk biomarkers (MPO, MMP-9, sE-selectin, sVCAM, sICAM, and PAI-1) were analyzed. Seven SNPs in the NPY gene were genotyped. The results of our study indicate that anthropometric measurements, clinical and metabolic markers, adipokines (leptin and resistin), and inflammatory and CVD risk biomarkers were generally elevated in the obese group. The exceptions to this finding included cholesterol, HDL-c, and adiponectin, which were lower in the obese group, and glucose, LDL-c, and MMP-9, which did not differ between the groups. Both rs16147 and rs16131 were associated with the risk of obesity, and the latter was also associated with insulin resistance, triacylglycerols, leptin, and HDL-c. Thus, we confirm the association of rs16147 with obesity, and we demonstrate for the first time the association of rs16131 with obesity and its possible impact on the early onset of metabolic syndrome features, mainly triacylglycerols, in children.     

Analysis of genetic variants of the poly(ADP-ribose) polymerase-1 gene in breast cancer in French patients

Previous studies have provided evidence for an association between exposure to high levels of air pollution and increased DNA damage in human sperm. In these studies DNA damage was measured using the sperm chromatin structure assay (SCSA) wherein the percentage of sperm with abnormal chromatin/fragmented DNA is determined and expressed as % DNA fragmentation index (%DFI). Here we extend these observations to address the following hypothesis: men who are homozygous null for glutathione-S-transferase M1 (GSTM1−) are less able to detoxify reactive metabolites of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) found in air pollution. Consequently they are more susceptible to the effects of air pollution on sperm chromatin. Using a longitudinal study design in which men provided semen samples during periods of both low (baseline) and episodically high air pollution, this study revealed a statistically significant association between GSTM1 null genotype and increased SCSA-defined %DFI (beta = 0.309; 95% CI: 0.129, 0.489). Furthermore, GSTM1 null men also showed higher %DFI in response to exposure to intermittent air pollution (beta = 0.487; 95% CI: 0.243, 0.731). This study thus provides novel evidence for a gene–environment interaction between GSTM1 and air pollution (presumably c-PAHs). The significance of the findings in this study with respect to fertility status is unknown. However, it is biologically plausible that increases in %DFI induced by such exposures could impact the risk of male sub/infertility, especially in men who naturally exhibit high levels of %DFI.     

Increased micronucleus frequency after oral administration of cadmium in dogs

Cadmium (Cd) is a toxic heavy metal that has been classified as a human carcinogen by the International Agency for Research on Cancer. The genotoxic effects of cadmium oxide (CdO) were investigated in cultured dog lymphocytes after a short-term oral CdO administration by the micronucleus (MN) test. The dogs were given 10 mg CdO/kg body weight per day for 3 and 28 d, respectively group I (n=7) and group II (n=6). Blood samples were collected at the beginning of feeding and at 4 and 29 d after Cd administration and cultured for 72 h. Whereas no significant increase in the MN frequency in group I was observed (p=0.398), a significant MN induction with CdO was found in group II (p=0.028) when compared with initial MN frequencies of dogs in both groups. Our results suggest that CdO might be directly and/or indirectly genotoxic after a monthly oral administration of CdO in dogs.     

Transferase S-glutathione class pi gene (GSTP1) polymorphism in thyroid cancer patients

INTRODUCTION: One of the potential genes which can increase the risk of cancer is GSTP1 gene. It encodes enzyme called glutathione S-transferase pi class, which is involved in the detoxification of a variety of potential carcinogenic compounds. Polymorphism in this gene can cause the amino acid substitution. This substitution, close to the substrate binding site, changes the enzymatic activity for particular substrates and subsequently increases the risk of carcinogenesis. The aim of this study was to evaluate the function of GSTP1 polymorphism in thyroid cancer and possible association between GSTP1 polymorphism and age at diagnosis. MATERIAL AND METHODS: 103 patients with differentiated thyroid cancer (DTC) and 53 individuals from control group were examined using PCR-RFLP. RESULTS: Statistically insignificant association of studied polymorphisms with thyroid cancer was observed. Comparison of allele frequency between cases and control groups revealed the presence of risk alleles. For the first polymorphism Ile OR = 1.257; 95% CI [0.792-1.997] (p = 0.332), and for the second one Val OR = 1.283; 95% CI [0.6260-2.631] (p = 0.495). The presence of Val/Val (c.313A>G) led to a significant earlier age of onset as compared with other genotypes (p < 0.05). Mean age at diagnosis for Val/Val genotype was 41.1 +/- 15.2, and for Ile/Val + Ile/Ile reached 48.9 +/- 13.2. There was no association between age and genotype for c.341C>T polymorphism. CONCLUSIONS: Statistically insignificant association of GSTP1 gene polymorphism with thyroid cancer was observed in studied group of patients. The Val/Val genotype for c.313A>G polymorphism led to earlier age of tumour diagnosis as compared with other genotypes.     

Effects of high frequency electromagnetic fields on micronucleus formation in CHO-K1 cells

To investigate the effects of high frequency electromagnetic fields (HFEMFs), we assessed the frequency of micronucleus (MN) formation induced by chromosomal breakage or inhibition of spindles during cell division in Chinese hamster ovary (CHO)-K1 cells, using the cytokinesis block micronucleus method. The MN frequency in cells in the inner, middle and outer wells of an annular culture plate was determined for the following four conditions: (1) CHO-K1 cells were exposed to a HFEMF for 18 h at average specific absorption rates (SARs) of 13, 39 and 50 W/kg with input power 7.8 W, and were compared with a sham-exposed control; (2) the cells were also exposed to a HFEMF at SARs of 78 and 100 W/kg with input power 13 W, and were compared with a sham-exposed control; (3) the cells were treated with bleomycin alone or with bleomycin followed by exposure to a HFEMF for 18 h at SARs of 25, 78 and 100 W/kg, and were compared with a bleomycin-treated positive control. The cells treated with bleomycin alone were compared with sham-exposed controls; and (4) As a high temperature control, CHO-K1 cells were incubated at 39 degrees C for 18 h. In study (1), the MN frequency of cells exposed to a HFEMF at a SAR of up to 50 W/kg was not different to that in sham-exposed cells. In study (2), there were statistically significant increases in the MN frequencies of cells in the middle and outer wells of the annular culture plate caused by exposure to a HFEMF at 100 and 78 W/kg, respectively. In study (3), the MN frequencies of cells in the middle (100 W/kg) and outer wells (78 W/kg) of the annular culture plate were statistically higher than that caused by bleomycin-treatment alone. In study (4), there was a statistically significant increase of MN frequency in the cells treated by heat at 39 degrees C.These results indicate that cells exposed to a HFEMF at a SAR of 78 W/kg and higher form MN more frequently than sham-exposed cells, while exposure to a HFEMF at up to 50 W/kg does not induce MN formation. In addition, a HFEMF at a SAR of 78 W/kg and higher may potentiate MN formation induced by bleomycin-treatment.     

On the frequency of copy number variants

MOTIVATION: Estimating the frequency distribution of copy number variants (CNVs) is an important aspect of the effort to characterize this new type of genetic variation. Currently, most studies report a strong skew toward low-frequency CNVs. In this article, our goal is to investigate the frequencies of CNVs. We employ a two-step procedure for the CNV frequency estimation process. We use family information a posteriori to select only the most reliable CNV regions, i.e. those showing high rates of Mendelian transmission. RESULTS: Our results suggest that the current skew toward low-frequency CNVs may not be representative of the true frequency distribution, but may be due, among other reasons, to the non-negligible false negative rates that characterize CNV detection methods. Moreover, false positives are also likely, as low-frequency CNVs are hard to detect with small sample sizes and technologies that are not ideally suited for their detection. Without appropriate validation methods, such as incorporation of biologically relevant information (for example, in our case, the transmission of heritable CNVs from parents to offspring), it is difficult to assess the validity of specific CNVs, and even harder to obtain reliable frequency estimates.     

Influence of apoA-V gene variants on postprandial triglyceride metabolism: impact of gender

Although apolipoprotein A-V (apoA-V) polymorphisms have been consistently associated with fasting triglyceride (TG) levels, their impact on postprandial lipemia remains relatively unknown. In this study, we investigate the impact of two common apoA-V polymorphisms (-1131 T>C and S19W) and apoA-V haplotypes on fasting and postprandial lipid metabolism in adults in the United Kingdom (n = 259). Compared with the wild-type TT, apoA-V -1131 TC heterozygotes had 15% (P = 0.057) and 21% (P = 0.002) higher fasting TG and postprandial TG area under the curve (AUC), respectively. Significant (P = 0.038) and nearly significant (P = 0.057) gender x genotype interactions were observed for fasting TG and TG AUC, with a greater impact of genotype in males. Lower HDL-cholesterol was associated with the rare TC genotype (P = 0.047). Significant linkage disequilibrium was found between the apoA-V -1131 T>C and the apoC-III 3238 C>G variants, with univariate analysis indicating an impact of this apoC-III single nucleotide polymorphism (SNP) on TG AUC (P = 0.015). However, in linear regression analysis, a significant independent association with TG AUC (P = 0.007) was only evident for the apoA-V -1131 T>C SNP, indicating a greater relative importance of the apoA-V genotype.     

Influence of ABCB1 genetic variants in breast cancer treatment outcomes

BackgroundTransportation of anticancer drugs such as anthracyclines across the membrane is regulated by P-glycoprotein encoded by the human multidrug resistance gene 1 (ABCB1). Polymorphisms in the ABCB1 gene (1236C > T, 2677G > T/A, 3435C > T) have been found to be associated with intrinsic and acquired cross resistance to these anticancer drugs. Therefore, the aim of this study is to evaluate the influence of ABCB1 gene polymorphisms in breast cancer treatment outcomes in terms of response and toxicity.MethodResponse to neo-adjuvant chemotherapy was evaluated in 100 patients while grade 2–4 toxicity was followed in 207 patients, who had undergone FEC/FAC chemotherapy. Genotyping for ABCB1 polymorphisms was done by PCR-RFLP. Chi square and logistic regression analyses were used to calculate Odd's ratio using SPSS ver 17.0. A meta analysis was also performed using Comprehensive Meta Analysis Ver 2.ResultsIn response evaluation, 1236C > T polymorphism was significantly associated with treatment response for CT genotype [OR = 5.17(1.3–20.2), P = 0.018] and in dominant model (CC vs CT + TT) [OR = 4.63(1.25–17.0), P = 0.021]. In the toxicity group, the T allele of 1236C>T was associated with grade 2–4 tocxicity [OR 1.48(1.00–2.20), P = 0.049] and the association was also significant in the recessive model [OR 1.88(1.05–3.39), P = 0.033]. For other two SNPs 2677G>T/A and 3435C>T no association was seen with either treatment response or grade 2–4 toxicity. In meta analysis, no overall association was found.ConclusionIn our study, significant association was seen for ABCB1 1236C>T polymorphism with treatment response. The meta analysis did not show overall association with treatment outcomes.