scholarly journals Validation of the in vitro comet assay for DNA cross-links and altered bases detection

2021 ◽  
Author(s):  
Damián Muruzabal ◽  
Julen Sanz-Serrano ◽  
Sylvie Sauvaigo ◽  
Bertrand Treillard ◽  
Ann-Karin Olsen ◽  
...  
Keyword(s):  
Comet Assay ◽  
Human Health Risk ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Vitro Assay ◽  
Cytotoxic Compound ◽  
Endonuclease Iii ◽  
Cross Links

AbstractMechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

scholarly journals A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone.

2002 ◽  
Vol 49 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Janusz Błasiak ◽  
Ewa Gloc ◽  
Mariusz Warszawski
Keyword(s):  
Dna Damage ◽  
Human Lymphocytes ◽  
Dna Glycosylase ◽  
Oxygen Species ◽  
Anthracycline Antibiotic ◽  
Strand Breaks ◽  
Endonuclease Iii ◽  
Normal Human ◽  
In Vitro Genotoxicity

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.

What can we Predict Before it's Implanted?

1985 ◽  
Vol 55 ◽  
Author(s):  
Donald F. Gibbons
Keyword(s):  
Biological Response ◽  
High Sensitivity ◽  
Biological Pathways ◽  
Surface Topology ◽  
Dystrophic Calcification ◽  
Vitro Assay ◽  
In Vivo Assays ◽  
Tissue Interface

ABSTRACTThe material factors which relate to the degradation and/or leaching of ions or molecules are described and the possible biological pathways which they may activate are described, i.e. cytotoxic, immune, tumor and nonspecific inflammatory response. Cytotoxicity is the only biological response which may be measured with high sensitivity by an in vitro assay prior to implantation. All other biological pathways require some degree of in vivo involvement. Three examples of biological response to material factors associated with devices which require evaluation by in vivo assays are discussed, namely: surface topology (texture), mechanically induced factors at the device/tissue interface caused by differences in compliance, and dystrophic calcification in connective tissue and vascular devices.

Differences in Repair of DNA Cross-links between Lymphocytes and Epithelial Tumor Cells from Colon Cancer Patients Measured In vitro with the Comet Assay

2009 ◽  
Vol 15 (17) ◽  
pp. 5466-5472 ◽  
Author(s):  
Mercedes Herrera ◽  
Gemma Dominguez ◽  
Jose M. Garcia ◽  
Cristina Peña ◽  
Carmen Jimenez ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Comet Assay ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Epithelial Tumor ◽  
Cross Links ◽  
Epithelial Tumor Cells

Imatinib (STI571) Induces DNA Damage in BCR/ABL-Expressing Leukemic Cells but Not in Normal Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4353-4353
Author(s):  
Janusz Blasiak ◽  
Jozef Drzewoski ◽  
Tomasz Poplawski ◽  
Agnieszka Czechowska
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Human Lymphocytes ◽  
Direct Interaction ◽  
K562 Cells ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Leukemic Cells ◽  
Drug Induced ◽  
Strand Breaks

Abstract Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets specifically the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.05 to 2 μM induced DNA damage in human leukemic K562 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the plasmid relaxation assay. K562 cells were unable to repair H2O2-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes.

Detection of alpha-methylacyl-CoA racemase (AMACR), a biomarker of prostate cancer, in patient blood samples using a nanoparticle electrochemical biosensor.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 41-41
Author(s):  
Matthew M. Cooney ◽  
Cheryl L. Thompson ◽  
Po-Yuan Lin ◽  
Kai-Lun Cheng ◽  
James D. McGuffin-Cawley ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
High Sensitivity ◽  
Intraepithelial Neoplasia ◽  
Detection Methods ◽  
Assay Method ◽  
Vitro Assay ◽  
Single Use ◽  
Detection And Diagnosis

41 Background: A promising prostate cancer biomarker, alpha-methylacyl-CoA racemase (AMACR), has demonstrated the ability to distinguish cancer from healthy and benign cells with high sensitivity and specificity. However the lack of a good clinical assay has limited its translation into the clinic. Here we report on the development of a single use disposable biosensor for AMACR detection. Methods: This is a very inexpensive, small, single-use disposable sensor that requires only a drop of plasma and connects to a portable device. The biosensor utilizes the reaction of pristanic acid with a substrate that includes AMACR to produce Trans-2,3-dehydropritanayl-CoA plus H2O2. The biosensor utilizes iridium oxide nanoparticle catalyst to oxidize the H2O2 produced in the above metabolic pathway. Thus the oxidation of H2O2 yields a measurable current to quantify AMACR in the sample. This is the first in vitro assay method for AMACR based on this reaction mechanism. Results: In our study including plasma from 9 healthy males, 10 patients with high grade prostatic intraepithelial neoplasia and 5 prostate cancer patients we show 100% accuracy in separating prostate cancer patients from controls as well as those with benign prostate conditions. Conclusions: Our data provides strong evidence for the ability of this biosensor to perform as a reliable assay for prostate cancer detection and diagnosis.

scholarly journals In Vitro Genotoxicity Assessment of Functional Ingredients: Betaine, Choline, and Taurine

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 339
Author(s):  
Julen Sanz-Serrano ◽  
Ariane Vettorazzi ◽  
Damian Muruzabal ◽  
Amaya Azqueta ◽  
Adela López de Cerain
Keyword(s):  
Micronucleus Test ◽  
Food Additives ◽  
Dna Glycosylase ◽  
World Health ◽  
Functional Ingredients ◽  
Endonuclease Iii ◽  
Health Organization ◽  
Tk6 Cells ◽  
In Vitro Genotoxicity

This article focuses on a complete in vitro genotoxicity assessment of three nutrients widely used as functional ingredients in the European market: betaine, choline, and taurine. The European Food Safety Authority (EFSA) tiered approach for food additives in concordance with the safety assessment of chemicals in food developed by Food and Agriculture Organization/World Health Organization (FAO/WHO) was followed; the miniaturized Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of Organization for Economic Co-operation and Development (OECD) 471), and the micronucleus test (OECD 487) in TK6 cells were performed. In addition, the in vitro standard and enzyme-modified (human 8-oxoguanine DNA glycosylase 1 (hOGG), endonuclease III (EndoIII), 3-alkyladenine DNA glycosylase (hAAG)) comet assay (S9−/S9+) was conducted in order to assess the potential premutagenic lesions in TK6 cells. None of the compounds produced any signs of genotoxicity in any of the conditions tested. This article increases the limited evidence available and complements the EFSA recommendations for the in vitro genotoxicity testing of nutrients.

scholarly journals In vitro assay for erythropoietin: erythroid colony formation in methyl cellulose used for the measurement of erythropoietin in plasma

Blood ◽  
1979 ◽  
Vol 53 (6) ◽  
pp. 1172-1181 ◽  
Author(s):  
P Haga ◽  
B Falkanger
Keyword(s):  
Colony Formation ◽  
Methyl Cellulose ◽  
High Sensitivity ◽  
In Vitro Assay ◽  
Target Cells ◽  
Newborn Mouse ◽  
Newborn Mice ◽  
Vitro Assay ◽  
Good Agreement

Erythroid colony formation in methyl cellulose has been used for the measurement of erythropoietin in plasma. Livers from newborn mice less than 24 hr old were found to provide convenient target cells. Newborn mouse liver contains a substantial number of erythroid colony-forming cells (CFU-e) that have a high sensitivity to erythropoietin, the dose-- response curve for erythropoietin reaching a plateau at 50 mU/ml. As little as 0.5 m/ml of the hormone is detectable. Removal of cells that adhered to glass prior to culturing doubled the number of colonies formed in the presence of erythropoietin. Addition of untreated plasmas that showed high erythropoietin titers in the exhypoxic polycythemic mice assay gave variable results. Some of the plasmas stimulated colony formation actively and in a linear fashion. However, the majority of the plasmas were toxic to the cultures. Dialyzing the plasmas for 3 days against distilled water effectively removed the toxicity. Results obtained with the method are in good agreement with the values found using the exhypoxic polycythemic mice assay.

Antigenotoxic potential of the fermentation broth produced by Paenibacillus polymyxa RNC-D in vitro

2021 ◽  
Author(s):  
Jaqueline Bianchi ◽  
Rafael Cavicchioli ◽  
Lauro T Kubota ◽  
Emanuel Carrilho ◽  
Cristina P de Sousa ◽  
...  
Keyword(s):  
Comet Assay ◽  
Micronucleus Test ◽  
Fermentation Broth ◽  
Paenibacillus Polymyxa ◽  
Dna Lesions ◽  
Methyl Methanesulfonate ◽  
Oxygen Species ◽  
Tnf Α ◽  
Antigenotoxic Effect

Aim: Evaluate the chemopreventive potential of the extract from P. polymyxa RNC-D. Methods: Concentrations of P. polymyxa RNC-D extract were tested in HepG2/C3A cells to assess their genotoxic (comet assay), mutagenic (micronucleus test) and antigenotoxic potential (comet assay) in vitro. Results: 400 and 40 μg/ml concentrations induced DNA lesions, whereas the 4 μg/ml induced a desmutagenic effect. Complementary tests indicated that the extract minimized the formation of reactive oxygen species induced by methyl methanesulfonate and normalized the loss of membrane potential. The quantification of cytokines indicated that TNF-α was immunostimulated by the extract. However, when administered in conjunction with the methyl methanesulfonate, the extract blocked the TNF-α release. Conclusion: The fermentation broth from P. polymyxa RNC-D showed an antigenotoxic effect, and thus the potential to be used as chemopreventive compound.

scholarly journals In vitro assay for erythropoietin: erythroid colony formation in methyl cellulose used for the measurement of erythropoietin in plasma

Blood ◽  
1979 ◽  
Vol 53 (6) ◽  
pp. 1172-1181 ◽  
Author(s):  
P Haga ◽  
B Falkanger
Keyword(s):  
Colony Formation ◽  
Methyl Cellulose ◽  
High Sensitivity ◽  
In Vitro Assay ◽  
Target Cells ◽  
Newborn Mouse ◽  
Newborn Mice ◽  
Vitro Assay ◽  
Good Agreement

Abstract Erythroid colony formation in methyl cellulose has been used for the measurement of erythropoietin in plasma. Livers from newborn mice less than 24 hr old were found to provide convenient target cells. Newborn mouse liver contains a substantial number of erythroid colony-forming cells (CFU-e) that have a high sensitivity to erythropoietin, the dose-- response curve for erythropoietin reaching a plateau at 50 mU/ml. As little as 0.5 m/ml of the hormone is detectable. Removal of cells that adhered to glass prior to culturing doubled the number of colonies formed in the presence of erythropoietin. Addition of untreated plasmas that showed high erythropoietin titers in the exhypoxic polycythemic mice assay gave variable results. Some of the plasmas stimulated colony formation actively and in a linear fashion. However, the majority of the plasmas were toxic to the cultures. Dialyzing the plasmas for 3 days against distilled water effectively removed the toxicity. Results obtained with the method are in good agreement with the values found using the exhypoxic polycythemic mice assay.

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