Single-Center Analysis of Essential Laboratory Testing in Patients with Newly Diagnosed Celiac Disease.

OBJECTIVE: To analyze laboratory testing results from pediatric patients newly diagnosed with celiac disease to determine the usefulness of each test derived from recommended guidelines. METHODS: Serological testing at the time of diagnosis from patients enrolled in our celiac disease registry from January 2018 through December 2021 was reviewed. The incidence of abnormal laboratory results, routinely obtained as per the recommendations of Snyder et al and our institution's Celiac Care Index, was assessed. Rates of abnormal laboratory values and estimated costs associated with these screening measures were analyzed. RESULTS: Our data demonstrated abnormalities in all serological testing obtained at celiac diagnosis. Hemoglobin, alanine aminotransferase, ferritin, iron, and vitamin D screening were found to be abnormal with notable frequency. Only 7% of patients had an abnormal thyroid-stimulating hormone and <0.1% had an abnormal free T4. Nonresponse to hepatitis B vaccination was prominent, with 69% of patients considered nonimmune. Screening protocols as currently outlined in our Celiac Care Index resulted in an estimated cost of approximately $320 000 during our study. CONCLUSIONS: Review of screening laboratory results at our center reveals that abnormal values for several recommended measures are uncommon. Thyroid screening was infrequently abnormal and the usefulness of screening for hepatitis B at diagnosis is uncertain. Similarly, our data suggest that iron deficiency screening may be condensed effectively into hemoglobin and ferritin testing, eliminating the need for initial iron studies. Decreasing baseline screening measures could safely decrease the burden of testing on patients and overall healthcare expenditures.

Determination of N7-glycidamide guanine adducts in human blood DNA following exposure to dietary acrylamide using liquid chromatography/tandem mass spectrometry.

RATIONALE: Acrylamide is classified as a probable human carcinogen that is metabolised to glycidamide, which can covalently bind to DNA. The aim of this study was to investigate the formation of N7-glycidamide guanine (N7-GA-Gua) adducts in human blood DNA following exposure to acrylamide present in carbohydrate-rich foods as part of the normal human diet. METHODS: Lymphocyte DNA was extracted from blood samples obtained from healthy human volunteers. Following thermal depurination of the DNA samples, N7-GA-Gua adducts were quantified using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method incorporating a stable isotope labelled internal standard. Estimated dietary acrylamide intake was recorded by completion of food frequency questionnaires for the 24 hours prior to volunteer blood donation. RESULTS: An LC/MS/MS method was validated with a limit of detection of 0.25 fmol and a lower limit of quantitation of 0.50 fmol on column. N7-GA-Gua adducts were detected in human blood DNA with the levels ranging between 0.3 to 6.3 adducts per 108 nucleotides. The acrylamide intake was calculated from the food frequency questionnaires ranging between 20.0 and 78.6 µg. CONCLUSIONS: Identification and quantification of N7-GA-Gua adducts in the blood DNA of healthy volunteers suggests that dietary acrylamide exposure may lead to the formation of DNA adducts. This important finding warrants further investigation to ascertain a correlation between environmental/dietary acrylamide exposure and levels of DNA adducts.

CD5-Positive Intravascular Large B-Cell Lymphoma in a Patient with Wilson's Disease: Case Report and Review of the Literature.

Intravascular large B-cell lymphoma (IVLBCL) is a rare extra-nodal B-cell lymphoma that proliferates within small/intermediate blood vessels and capillaries while sparing large blood vessels and organ parenchyma. Clinical presentation is highly variable and may include B symptoms, neurological deficits, and/or cutaneous findings. The diagnosis of IVLBCL is difficult due to multiorgan involvement and nonspecific symptoms. We describe the case of a 68-year-old male who presented with progressive weakness, confusion, and falls. He had a past medical history of liver cirrhosis secondary to Wilson's disease. Physical exam and laboratory results revealed a lethargic man with jaundice, hepatic encephalopathy, and abnormal liver/kidney function tests. He expired after a short hospital course in the setting of hepatic and renal failure. Postmortem examination revealed large neoplastic lymphoid cells involving multiple organ blood vessels; however skin and neurologic involvement was absent. The neoplastic cells demonstrated B-cells positive for CD5, rendering a diagnosis of IVLBCL. Our case represents the occurrence of IVLBCL with CD5-positivity in a patient with Wilson's disease, diagnosed at autopsy demonstrating the challenging nature of diagnosing IVLBCL.

Risks to human and animal health related to the presence of moniliformin in food and feed.

Moniliformin (MON) is a mycotoxin with low molecular weight primarily produced by Fusarium fungi and occurring predominantly in cereal grains. Following a request of the European Commission, the CONTAM Panel assessed the risk of MON to human and animal health related to its presence in food and feed. The limited information available on toxicity and on toxicokinetics in experimental and farm animals indicated haematotoxicity and cardiotoxicity as major adverse health effects of MON. MON causes chromosome aberrations in vitro but no in vivo genotoxicity data and no carcinogenicity data were identified. Due to the limitations in the available toxicity data, human acute or chronic health-based guidance values (HBGV) could not be established. The margin of exposure (MOE) between the no-observed-adverse-effect level (NOAEL) of 6.0 mg/kg body weight (bw) for cardiotoxicity from a subacute study in rats and the acute upper bound (UB) dietary exposure estimates ranged between 4,000 and 73,000. The MOE between the lowest benchmark dose lower confidence limit (for a 5% response - BMDL05) of 0.20 mg MON/kg bw per day for haematological hazards from a 28-day study in pigs and the chronic dietary human exposure estimates ranged between 370 and 5,000,000 for chronic dietary exposures. These MOEs indicate a low risk for human health but were associated with high uncertainty. The toxicity data available for poultry, pigs, and mink indicated a low or even negligible risk for these animals from exposure to MON in feed at the estimated exposure levels under current feeding practices. Assuming similar or lower sensitivity as for pigs, the CONTAM Panel considered a low or even negligible risk for the other animal species for which no toxicity data suitable for hazard characterisation were identified. Additional toxicity studies are needed and depending on their outcome, the collection of more occurrence data on MON in food and feed is recommended to enable a comprehensive human risk assessment.

Risk to human and animal health related to the presence of 4,15-diacetoxyscirpenol in food and feed.

4,15-Diacetoxyscirpenol (DAS) is a mycotoxin primarily produced by Fusarium fungi and occurring predominantly in cereal grains. As requested by the European Commission, the EFSA Panel on Contaminants in the Food Chain (CONTAM) assessed the risk of DAS to human and animal health related to its presence in food and feed. Very limited information was available on toxicity and on toxicokinetics in experimental and farm animals. Due to the limitations in the available data set, human acute and chronic health-based guidance values (HBGV) were established based on data obtained in clinical trials of DAS as an anticancer agent (anguidine) after intravenous administration to cancer patients. The CONTAM Panel considered these data as informative for the hazard characterisation of DAS after oral exposure. The main adverse effects after acute and repeated exposure were emesis, with a no-observed-adverse-effect level (NOAEL) of 32 µg DAS/kg body weight (bw), and haematotoxicity, with a NOAEL of 65 µg DAS/kg bw, respectively. An acute reference dose (ARfD) of 3.2 µg DAS/kg bw and a tolerable daily intake (TDI) of 0.65 µg DAS/kg bw were established. Based on over 15,000 occurrence data, the highest acute and chronic dietary exposures were estimated to be 0.8 and 0.49 µg DAS/kg bw per day, respectively, and were not of health concern for humans. The limited information for poultry, pigs and dogs indicated a low risk for these animals at the estimated DAS exposure levels under current feeding practices, with the possible exception of fattening chicken. Assuming similar or lower sensitivity than for poultry, the risk was considered overall low for other farm and companion animal species for which no toxicity data were available. In consideration of the similarities of several trichothecenes and the likelihood of co-exposure via food and feed, it could be appropriate to perform a cumulative risk assessment for this group of substances.

Maternal diet during pregnancy and micronuclei frequency in peripheral blood T lymphocytes in mothers and newborns (Rhea cohort, Crete).

PURPOSE: The study assessed whether diet and adherence to cancer prevention guidelines during pregnancy were associated with micronucleus (MN) frequency in mothers and newborns. MN is biomarkers of early genetic effects that have been associated with cancer risk in adults. METHODS: A total of 188 mothers and 200 newborns from the Rhea cohort (Greece) were included in the study. At early-mid pregnancy, we conducted personal interviews and a validated food frequency questionnaire was completed. With this information, we constructed a score reflecting adherence to the World Cancer Research Fund/American Institute for Cancer Research cancer prevention guidelines on diet, physical activity and body fatness. At delivery, maternal and/or cord blood was collected to measure DNA and hemoglobin adducts of dietary origin and frequencies of MN in binucleated and mononucleated T lymphocytes (MNBN and MNMONO). RESULTS: In mothers, higher levels of red meat consumption were associated with increased MNBN frequency [2nd tertile IRR = 1.34 (1.00, 1.80), 3rd tertile IRR = 1.33 (0.96, 1.85)] and MNMONO frequency [2nd tertile IRR = 1.53 (0.84, 2.77), 3rd tertile IRR = 2.69 (1.44, 5.05)]. The opposite trend was observed for MNBN in newborns [2nd tertile IRR = 0.64 (0.44, 0.94), 3rd tertile IRR = 0.68 (0.46, 1.01)], and no association was observed with MNMONO. Increased MN frequency in pregnant women with high red meat consumption is consistent with previous knowledge. CONCLUSIONS: Our results also suggest exposure to genotoxics during pregnancy might affect differently mothers and newborns. The predictive value of MN as biomarker for childhood cancer, rather than adulthood, remains unclear. With few exceptions, the association between maternal carcinogenic exposures during pregnancy and childhood cancer or early biologic effect biomarkers remains poorly understood.

Analysis of the Cytomorphological Features in Atypical Urine Specimens following Application of The Paris System for Reporting Urinary Cytology.

BACKGROUND: This study investigates the use of The Paris System (TPS) for Reporting Urinary Cytopathology and examines the performance of individual and combined morphological features in atypical urine cytologies. METHODS: We reviewed 118 atypical cytologies with subsequent bladder biopsies for the presence of several morphological features and reclassified them into Paris System categories. The sensitivity and specificity of individual and combined features were calculated along with the risk of malignancy. RESULTS: An elevated nuclear-to-cytoplasmic ratio was only predictive of malignancy if seen in single cells, while irregular nuclear borders, hyperchromasia, and coarse granular chromatin were predictive in single cells and in groups. Identification of coarse chromatin alone yielded a malignancy risk comparable to 2-feature combinations. The use of TPS criteria identified the specimens at a higher risk of malignancy. CONCLUSION: Our findings support the use of TPS criteria, suggesting that the presence of coarse chromatin is more specific than other individual features, and confirming that cytologic atypia is more worrisome in single cells than in groups.

The impact of subdividing the "atypical" category for urinary cytology on patient management.

INTRODUCTION: The purpose of the study is to determine the impact of subdividing the "atypical" cytology interpretation into two groups: Atypical urothelial cells of uncertain significance (AUC-US) and Atypical urothelial cells suspicious for high-grade urothelial carcinoma (AUC-H/SHGUC), on management of patients with no prior history of UC. MATERIALS AND METHOD: This is a retrospective study of "atypical" urine cytology with subsequent tissue examination occurring within six months. Cytology reports with "atypical" interpretation were reclassified into AUS-UC and AUC-H based on morphologic features identified by the Johns Hopkins system and the Paris system for urine cytology. Follow-up and categorical outcomes were compared between the reclassified AUC-US and AUC-H groups. RESULTS: There was no significant difference (P < 0.4539) in the rate of cytology follow-up, the follow-up cytology result (P < 0.1845), or time between follow-up cytologies (P < 0.0869) between the reclassified atypical group of AUC-H and AUC-US. There was a significant association (P < 0.0001) of rate of malignancy with the reclassified AUC-H (87.18%) compared to the AUC-US (58.68%) groups. CONCLUSION: There was no difference in follow-up between the AUC-H and AUC-US, however there was a difference in the rates of malignancy in the two groups. The AUC-H group is similar to the SHGUC group of the Paris system and can be considered as such, whereas the AUC-US group should continue to be considered atypical. We conclude that reclassification of the "atypical" category into AUC-US and AUC-H/SHGUC can reduce the rate of atypia and help in focused follow-up and targeted management. Diagn. Cytopathol. 2016;44:477-482. © 2016 Wiley Periodicals, Inc.

Antitumour benzothiazoles. Part 32: DNA adducts and double strand breaks correlate with activity; synthesis of 5F203 hydrogels for local delivery.

Potent, selective antitumour AhR ligands 5F 203 and GW 610 are bioactivated by CYPs 1A1 and 2W1. Herein we reason that DNA adducts' generation resulting in lethal DNA double strand breaks (DSBs) underlies benzothiazoles' activity. Treatment of sensitive carcinoma cell lines with GW 610 generated co-eluting DNA adducts (R(2)>0.7). Time-dependent appearance of γ-H2AX foci revealed subsequent DNA double strand breaks. Propensity for systemic toxicity of benzothiazoles steered development of prodrugs' hydrogels for localised delivery. Clinical applications of targeted therapies include prevention or treatment of recurrent disease after surgical resection of solid tumours. In vitro evaluation of 5F 203 prodrugs' activity demonstrated nanomolar potency against MCF-7 breast and IGROV-1 ovarian carcinoma cell lines.

An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride.

The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.

Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: the NewGeneris cohort.

BACKGROUND: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development. OBJECTIVES: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored. METHODS: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe. RESULTS: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure-outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG-DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN frequency. Polymorphisms in EPHX1/2 and CYP2E1 were associated with MNBN. CONCLUSION: We measured in utero exposure to selected environmental carcinogens and circulating hormonally acting factors and detected associations with MN frequency in newborns circulating T lymphocytes. The results highlight mechanisms that may contribute to carcinogen-induced leukemia and require further research.

Bulky dna adducts in cord blood, maternal fruit-and-vegetable consumption, and birth weight in a European mother-child study (NewGeneris).

BACKGROUND: Tobacco-smoke, airborne, and dietary exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with reduced prenatal growth. Evidence from biomarker-based studies of low-exposed populations is limited. Bulky DNA adducts in cord blood reflect the prenatal effective dose to several genotoxic agents including PAHs. OBJECTIVES: We estimated the association between bulky DNA adduct levels and birth weight in a multicenter study and examined modification of this association by maternal intake of fruits and vegetables during pregnancy. METHODS: Pregnant women from Denmark, England, Greece, Norway, and Spain were recruited in 2006-2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires. RESULTS: Adduct levels in maternal and cord blood samples were similar and positively correlated (median, 12.1 vs. 11.4 adducts in 108 nucleotides; Spearman rank correlation coefficient = 0.66, p < 0.001). Cord blood adduct levels were negatively associated with birth weight, with an estimated difference in mean birth weight of -129 g (95% CI: -233, -25 g) for infants in the highest versus lowest tertile of adducts. The negative association with birth weight was limited to births in Norway, Denmark, and England, the countries with the lowest adduct levels, and was more pronounced in births to mothers with low intake of fruits and vegetables (-248 g; 95% CI: -405, -92 g) compared with those with high intake (-58 g; 95% CI: -206, 90 g). CONCLUSIONS: Maternal exposure to genotoxic agents that induce the formation of bulky DNA adducts may affect intrauterine growth. Maternal fruit and vegetable consumption may be protective.

Brain metastases from papillary thyroid carcinomas.

Brain metastasis from papillary thyroid carcinoma (PTC) is extremely rare and carries a poor prognosis. We report nine cases (five females and four males) of brain metastasis of PTC. The age of patients ranged from 46 to 87 years old. The patients presented with nonspecific symptoms such as headaches. Brain metastasis was the first clinical presentation in three of nine patients; two of which had the aggressive tall cell variant of PTC. Six patients had prior history of PTC (four classic, one oncocytic variant, and one columnar cell variant) for 2 to 17 years with a median of 12 years. Gross total resection of brain metastasis was achieved for eight of our patients. Eight patients were treated with radioactive iodine. The median follow-up time was 12 months, ranging from 1 month to 4 years. Three patients died of their disease in 6 months, 21 months and 4 years, respectively after their first presentation of brain metastasis. It seems that these rare aggressive variants of PTC, such as tall cell variant, not only have higher propensity to develop brain metastasis, but also more frequently present with brain metastasis as their first clinical presentation than classic PTC. Furthermore, patients with PTC can develop brain metastasis even after many years.

Central nervous system lymphoma in immunocompetent patients: the North Shore-Long Island Jewish Health System experience.

Of the 74 immunocompetent patients diagnosed between July 2004 and June 2011 at the North Shore University Hospital and Long Island Jewish Medical Center with primary central nervous system lymphoma, 71 (95.9%) had diffuse large B-cell lymphomas (DLBCL). The median patient age was 68 years (range: 19-87 years) with a slight male preponderance (1.1:1). The overall median survival time was 21 months. For patients older than 70 years, the median survival time was 8 months while for those 70 years or younger, the median survival time was 27 months (p<0.01). Female patients had a worse prognosis than male patients (p<0.05, median survival time, 17 months compared to 23 months). We had enough data from 52 of these 71 patients to define the lymphomas as either germinal center B-cell-like (GCB) or activated B-cell-like (ABC) DLBCL. Of these 52 patients, 42 (80.8%) had ABC DLBCL while only 10 (19.2%) had GCB DLBCL. The patients in the GCB subgroup seemed to survive longer than the patients in the ABC subgroup, although the difference did not reach statistical significance. No statistically significant difference in overall survival was seen between patients with BCL-6 positive or negative DLBCL; or between patients with BCL-2 positive or negative DLBCL.

Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. RESULTS: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (rp 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). INNOVATION: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. CONCLUSION: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.

Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro.

Although some types of carbon nanotubes (CNTs) have been described to induce mesothelioma in rodents and genotoxic effects in various cell systems, there are few previous studies on the genotoxicity of CNTs in mesothelial cells. Here, we examined in vitro DNA damage induction by short multi-wall CNTs (MWCNTs; 10-30 nm × 1-2 µm) and single-wall CNTs (SWCNTs; >50% SWCNTs, ~40% other CNTs; <2 nm × 1-5 µm) in human mesothelial (MeT-5A) cells and bronchial epithelial (BEAS 2B) cells, using the single cell gel electrophoresis (comet) assay and the immunoslot blot assay for the detection of malondialdehyde (M1dG) DNA adducts. In BEAS 2B cells, we also studied the induction of micronuclei (MN) by the CNTs using the cytokinesis-block method. The cells were exposed to the CNTs (5-200 µg/cm(2), corresponding to 19-760 µg/ml) for 24 and 48h in the comet assay and for 48 and 72 h in the MN and M1dG assays. Transmission electron microscopy (TEM) showed more MWCNT fibres and SWCNT clusters in BEAS 2B than MeT-5A cells, but no significant differences were seen in intracellular dose expressed as area of SWCNT clusters between TEM sections of the cell lines. In MeT-5A cells, both CNTs caused a dose-dependent induction of DNA damage (% DNA in comet tail) in the 48-h treatment and SWCNTs additionally in the 24-h treatment, with a statistically significant increase at 40 µg/cm(2) of SWCNTs and (after 48 h) 80 µg/cm(2) of both CNTs. SWCNTs also elevated the level of M1dG DNA adducts at 1, 5, 10 and 40 µg/cm(2) after the 48-h treatment, but both CNTs decreased M1dG adduct level at several doses after the 72-h treatment. In BEAS 2B cells, SWCNTs induced a statistically significant increase in DNA damage at 80 and 120 µg/cm(2) after the 24-h treatment and in M1dG adduct level at 5 µg/cm(2) after 48 h and 10 and 40 µg/cm(2) after 72 h; MWCNTs did not affect the level of DNA damage but produced a decrease in M1dG adducts in the 72-h treatment. The CNTs did not affect the level of MN. In conclusion, MWCNTs and SWCNTs induced DNA damage in MeT-5A cells but showed a lower (SWCNTs) or no (MWCNTs) effect in BEAS 2B cells, suggesting that MeT-5A cells were more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, despite the fact that more CNT fibres or clusters were seen in BEAS 2B than MeT-5A cells. M1dG DNA adducts were induced by SWCNTs but decreased after a 3-day exposure to MWCNTs and (in MeT-5A cells) SWCNTs, indicating that CNTs may lead to alterations in oxidative effects within the cells. Neither of the CNTs was able to produce chromosomal damage (MN).

Urinary benzene biomarkers and DNA methylation in Bulgarian petrochemical workers: study findings and comparison of linear and beta regression models.

Chronic occupational exposure to benzene is associated with an increased risk of hematological malignancies such as acute myeloid leukemia (AML), but the underlying mechanisms are still unclear. The main objective of this study was to investigate the association between benzene exposure and DNA methylation, both in repeated elements and candidate genes, in a population of 158 Bulgarian petrochemical workers and 50 unexposed office workers. Exposure assessment included personal monitoring of airborne benzene at work and urinary biomarkers of benzene metabolism (S-phenylmercapturic acid [SPMA] and trans,trans-muconic acid [t,t-MA]) at the end of the work-shift. The median levels of airborne benzene, SPMA and t,t-MA in workers were 0.46 ppm, 15.5 µg/L and 711 µg/L respectively, and exposure levels were significantly lower in the controls. Repeated-element DNA methylation was measured in Alu and LINE-1, and gene-specific methylation in MAGE and p15. DNA methylation levels were not significantly different between exposed workers and controls (P>0.05). Both ordinary least squares (OLS) and beta-regression models were used to estimate benzene-methylation associations. Beta-regression showed better model specification, as reflected in improved coefficient of determination (pseudo R(2)) and Akaike's information criterion (AIC). In beta-regression, we found statistically significant reductions in LINE-1 (-0.15%, P<0.01) and p15 (-0.096%, P<0.01) mean methylation levels with each interquartile range (IQR) increase in SPMA. This study showed statistically significant but weak associations of LINE-1 and p15 hypomethylation with SPMA in Bulgarian petrochemical workers. We showed that beta-regression is more appropriate than OLS regression for fitting methylation data.

Detection of acetaldehyde derived N(2)-ethyl-2'-deoxyguanosine in human leukocyte DNA following alcohol consumption.

Epidemiological studies have shown an association between alcohol (ethanol) consumption and increased cancer risk. The effect of alcohol consumption on the levels and persistence of N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) formed by acetaldehyde, the oxidative metabolite of ethanol, in human leukocyte DNA was investigated. DNA was isolated from venous blood samples obtained from 30 male non-smoking individuals before consumption of alcohol (0h) and subsequently at 3-5h following the consumption of 150mL of vodka (containing 42% pure ethanol). Additional samples were collected 24h and 48h post-alcohol consumption. The levels of N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) in the DNA were determined following reduction of N(2)-ethylidene-dG with sodium cyanoborohydride using a liquid chromatography-tandem mass spectrometry selected reaction monitoring method. A slight time-dependent trend showing an increase and decrease in the levels of N(2)-ethyl-dG was observed following consumption of alcohol compared to time 0h, however, the differences were not statistically significant. The average levels of N(2)-ethyl-dG observed at 0h, 3-5h, 24h and 48h time points following ingestion of alcohol were 34.6±21.9, 35.1±21.0, 36.8±20.7 and 35.6±21.1 per 10(8) 2'-deoxynucleosides, respectively. In conclusion, alcohol consumption that could be encountered under social drinking conditions, does not significantly alter the levels of the acetaldehyde derived DNA adduct, N(2)-ethyl-dG in human leukocyte DNA from healthy individuals.

Glioblastoma with PNET-like components has a higher frequency of isocitrate dehydrogenase 1 (IDH1) mutation and likely a better prognosis than primary glioblastoma.

Glioblastoma with primitive neuroectodermal tumor-like components (GBM-PNET), a rare variant of glioblastoma, poses both diagnostic and therapeutic challenges. Ten patients with GBM-PNET were investigated with a median age of 51.5 years and the male to female ratio of 4:1. The majority of patients (7 out of 10) showed ring-enhancing lesions on magnetic resonance imaging (MRI), which is classic for GBMs. Restricted diffusion was noted in 7 cases where diffusion weighted imaging (DWI) was performed, which correlates with the presence of PNET-like components. CD56 and vimentin immunostaining made the diagnosis of GBM-PNET much easier. Vimentin strongly and diffusely highlighted the astrocytic components and was negative in PNET-like components, while CD56 was strongly and diffusely positive in both astrocytic and PNET-like components. Seven out of 9 cases were positive for p53 in both astrocytic and PNET-like components. Two out of 8 cases harbored isocitrate dehydrogenase 1 (IDH1) R132H mutation, while IDH2 R172 mutations were not identified. Three out of 10 patients had a median survival time of 17 months while the two patients, whose tumor carried IDH1 mutation, were still alive after 15 and 31 months of follow-up. Compared to primary GBMs, GBM-PNETs might have a better prognosis. Further large scale studies are necessary to confirm this observation.

Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adduct formation in P450 reductase conditional null mice.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were ∼3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average ∼2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was ∼8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.