Evaluation of staining techniques on the results of micronucleus in exfoliated oral mucosa cells

Micronuclei (MN) in exfoliated epithelial cells are widely used as biomarkers of cancer risk in humans. MN are classified as biomarkers of the break age and loss of chromosomes. They are small, extra nuclear bodies that arise in dividing cells from centric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase. Buccal mucosa cells have been used in biomonitoring exposed populations because these cells are in the direct route of exposure to ingested pollutant, are capable of metabolizing proximate carcinogens to reactive chemicals, and are easily and rapidly collected by brushing the buccal mucosa. The objective of the present study was to further investigate if, and to what extent, different stains have an effect on the results of micronuclei studies in exfoliated cells. These techniques are: Papanicolaou (PAP), Modified Papanicolaou, May-Grünwald Giemsa (MGG), Giemsa, Harris’s Hematoxylin, Feulgen with Fast Green counterstain and Feulgen without counterstain.

XRCC3 THR241MET polymorphism influence on nuclear buds frequency in exposed to formaldehyde

Formaldehyde (FA), also known as formalin, formal and methyl aldehydes, is a colorless, flammable, strong-smelling gas. It has an important application in embalming tissues and that result in exposures for workers in the pathology anatomy laboratories and mortuaries. Occupational exposure to FA has been shown to induce nasopharyngeal cancer and has been classified as carcinogenic to humans (group 1) on the basis of sufficient evidence in humans and sufficient evidence in experimental animals. Manifold in vitro studies clearly indicated that FA is genotoxic. FA induced various genotoxic effects in proliferating cultured mammalian cells. The cytokinesis-block micronucleus (CBMN) assay was originally developped as an ideal system form easuring micronucleus (MN), however it can also be used to measure nucleoplasmic bridges (NBP) and nuclear buds (NBUD). Over the past decade another unique mechanism of micronucleus formation, known as nuclear budding has emerged. NBUDS is considered as a marker of gene amplification and/or altered gene dosage because the nuclear budding process is the mechanism by which cells removed amplified and/excess DNA.

Formaldehyde's genotoxicity effects in pathology anatomy technologists and medical pathologists

Formaldehyde (FA) had been considered to be carcinogenic by the International Agency for Research on Cancer (group1), on the basis of sufficient evidence both in humans and in experimental animals, making it a subject of major environmental concern, especially in the occupational context. Manifold in vitro studies clearly indicated that FA is genotoxic, inducing various genotoxic effects in proliferating cultured mammalian cells. Cytokinesis-blocked micronucleus (CBMN) assay is used extensively in molecular epidemiology, and the chromosomal alterations most reported and studied by the CBMN are: micronucleus (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUDs). The pathology anatomy laboratories are work places that manipulate routinely FA and pathology anatomy technologists and pathologists contact daily with this chemical compound particularly in the macroscopic exam and grossing procedures. The aim of this study was to identify genotoxicity biomarkers in the set workers groups, such as micronucleus (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUD) in peripheral blood lymphocytes.

Occupational exposure to formaldehyde: effects of years of exposure in the frequency of micronucleus

Formaldehyde: an important industrial compound used in the manufacture of synthetic resins and chemical compounds such as lubricants and adhesives; also applied as a disinfectant, preservative and in cosmetics productions; relevant workplace exposure to FA also occurs in anatomy, pathology and in mortuaries; classified by IARC as carcinogenic to humans (Group 1), based on sufficient evidence in humans and experimental animals; manifold in vitro studies indicated that FA can induce genotoxic effects in proliferating cultured mammalian cells. Aim of the study: to evaluate if years of exposure induced a genotoxic biomarkers increase, namely MN in lymphocytes and buccal cells, in workers occupationally exposed to FA (factory and pathology anatomy laboratory).

Interaction of formaldehyde and tobacco smoking in the frequency of micronucleus and the XRCC3 Thr241Met polymorphism

Formaldehyde is classified by IARC as carcinogenic to humans (nasopharyngeal cancer). Tobacco smoke has been epidemiologically associated to a higher risk of development of cancer, especially in the oral cavity, larynx and lungs, as these are places of direct contact with many carcinogenic tobacco’s compounds. XRCC3 is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks (Thr241Met polymorphism). The aim of the study is to determine whether there is an in vivo association between genetic polymorphism of the gene XRCC3 and the frequency of genotoxicity biomarkers in subjects exposed or not to formaldehyde and with or without tobacco consumption.

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

The influence of genetic polymorphisms in XRCC3 and ADH5 genes on the frequency of genotoxicity biomarkers in workers exposed to formaldehyde

The International Agency for Research on Cancer classified formaldehyde as carcinogenic to humans because there is “sufficient epidemiological evidence that it causes nasopharyngeal cancer in humans”. Genes involved in DNA repair and maintenance of genome integrity are critically involved in protecting against mutations that lead to cancer and/or inherited genetic disease. Association studies have recently provided evidence for a link between DNA repair polymorphisms and micronucleus (MN) induction. We used the cytokinesis-block micronucleus (CBMN assay) in peripheral lymphocytes and MN test in buccal cells to investigate the effects of XRCC3 Thr241Met, ADH5 Val309Ile, and Asp353Glu polymorphisms on the frequency of genotoxicity biomarkers in individuals occupationally exposed to formaldehyde (n = 54) and unexposed workers (n = 82). XRCC3 participates in DNA double-strand break/recombination repair, while ADH5 is an important component of cellular metabolism for the elimination of formaldehyde. Exposed workers had significantly higher frequencies (P < 0.01) than controls for all genotoxicity biomarkers evaluated in this study. Moreover, there were significant associations between XRCC3 genotypes and nuclear buds, namely XRCC3 Met/Met (OR = 3.975, CI 1.053–14.998, P = 0.042) and XRCC3 Thr/Met (OR = 5.632, CI 1.673–18.961, P = 0.005) in comparison with XRCC3 Thr/Thr. ADH5 polymorphisms did not show significant effects. This study highlights the importance of integrating genotoxicity biomarkers and genetic polymorphisms in human biomonitoring studies.

Application of alkaline comet assay in human biomonitoring for genotoxicity: a study on occupational exposure to cytostatics

The use of cytostatics drugs in anticancer therapy is increasing. Health care workers can be occupationally exposed to these drugs classified as carcinogenic, mutagenic or teratogenic. Cytostatics drugs are a heterogeneous group of chemicals widely used in the treatment of cancer, nevertheless have been proved to be also mutagens, carcinogens and teratogens. Workers may be exposed to this drug, being in the hospital settings the main focus dwelled upon the pharmacy, and nursing personnel. Alkaline comet assay is one of the most promising short-term genotoxicity assays for human risk assessment, being recommended to monitor populations chronically exposed to genotoxic agents. DNA glycosylase (OGG1) represents the main mechanism of protecting the integrity of the human DNA with respect to 8-OHdG, the most well studied biomarker of oxidative damage.

Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata

The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.

Genotoxic assessment in different exposure groups working with antineoplastic agents

Antioneoplastic drugs are widely used in treatment of cancer, and several studies suggest acute and long-term effects associated to antineoplastic drug exposures, namely associating workplace exposure with health effects. Cytokinesis blocked micronucleus (CBMN) assay is one promising short-term genotoxicity assays for human risk assessment and their combination is recommended to monitor populations chronically exposed to genotoxic agents. The aim of this investigation is the genotoxicity assessment in different professionals that handle cytostatics drugs. This research is case-control blinded study constituted by 46 non-exposed subjects and 44 workers that handle antineoplastic drugs, such as pharmacists, pharmacy technicians, and nurses. It was found statistically significant increases in the genotoxicity biomarkers in exposed comparising with controls (p<0.05). The findings address the need for regular biomonitoring of personnel occupationally exposed to these drugs, confirming to an enhanced health risk assessment.

Anti-leishmania igA immunoenzymatic assay in mucocutaneous leishmaniasis (Preliminary report)

The Authors describe an anti-Leishmania IgA-ELISA assay in mucocutaneous leishmaniasis. Increased titers were found in leishmaniasis patients, mainly in the first and second year of infection and in deep mycoses patients showing either mucosal involvement or widespread disease.

An immunohistochemic assay to localize leptospires in tissue specimens

An Immunoperoxidase technique for identification of leptospires in formalin fixed, paraffin embedded kidney sections is presented, using peroxidase-antiperoxidase complex. The anti-leptospiral antibody was raised in rabbit. Possible applications of this technique are discussed.

Enzyme linked immunosorbent assay: determination of anti-adenovirus antibodies in an infant population

In order to define an accurate assay for anti-adenovirus antibody detection, a recently developed ELISA was compared with IFA and CF. On 58 sera, the ELISA was more sensitive than both CF and IFA, which showed relative sensitivities of 63% and 94%, respectively. It was not possible to determine the exact specificity of the tests because of the lack of a gold standard. Furthermore, the ELISA was used to define the prevalence of adenovirus antibodies in 116 infants between 1 and 24 months old (mean 7.28). The data showed that maternal antibodies waned by the age of 5 to 6 months and that more than 80% of the children had been infected by adenoviruses by the age of 10 months.

Screening the mutagenic activities of coomonly used antiparasite drugs by the Simultest, a simplified Salmonella/microsome plate incorporation assay

The mutagenic activities of 16 anti-parasite drugs were screened by the Simultest in both qualitative (spot test) and quantitative (plate incorporation) assays with a Salmonella typhimurium pool composed by the indicator strains TA97, TA 98, TA100 and TA102. Four anti Chagas' disease drugs (nifurtimox, benznidazole, CL 64,855, and MK 436) and two anti-amebae drugs (metronidazole and tinidazole) gave positive results in qualitative tests and incorporation of rat liver microsomes did not alter the results. Comparative dose response curves of the mutagenic activities of CL 64,855, metronidazole and benznidazole obtained by the simultest and by individual Salmonella indicator strains demonstrated that both approachs have similar sensitivities. The results corroborate the validity of the Simultest, as a simplified, fast and economic version of the Ames test in preliminary screening of potential mutagenic drugs.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.