Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/%28SICI%291098-2280%282000%2935%3A3%3C206%3A%3AAID-EM8%3E3.0.CO%3B2-J

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Suitable labels are at the core of Luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)--inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistry--as in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.

/pdf/quantum-dots-versus-organic-dyes-as-fluorescent-labels-50cbzj94q6.pdf

Quantum dots versus organic dyes as fluorescent labels

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.

/pdf/drug-delivery-and-nanoparticles-applications-and-hazards-3sdw6khbk9.pdf

Drug delivery and nanoparticles:applications and hazards

As the environment preservation gradually becomes a matter of major social concern and more strict legislation is being imposed on effluent discharge, more effective processes are required to deal with non-readily biodegradable and toxic pollutants. Synthetic organic dyes in industrial effluents cannot be destroyed in conventional wastewater treatment and consequently, an urgent challenge is the development of new environmentally benign technologies able to mineralize completely these non-biodegradable compounds. This review aims to increase the knowledge on the electrochemical methods used at lab and pilot plant scale to decontaminate synthetic and real effluents containing dyes, considering the period from 2009 to 2013, as an update of our previous review up to 2008. Fundamentals and main applications of electrochemical advanced oxidation processes and the other electrochemical approaches are described. Typical methods such as electrocoagulation, electrochemical reduction, electrochemical oxidation and indirect electro-oxidation with active chlorine species are discussed. Recent advances on electrocatalysis related to the nature of anode material to generate strong heterogeneous OH as mediated oxidant of dyes in electrochemical oxidation are extensively examined. The fast destruction of dyestuffs mediated with electrogenerated active chlorine is analyzed. Electro-Fenton and photo-assisted electrochemical methods like photoelectrocatalysis and photoelectro-Fenton, which destroy dyes by heterogeneous OH and/or homogeneous OH produced in the solution bulk, are described. Current advantages of the exposition of effluents to sunlight in the emerging photo-assisted procedures of solar photoelectrocatalysis and solar photoelectro-Fenton are detailed. The characteristics of novel combined methods involving photocatalysis, adsorption, nanofiltration, microwaves and ultrasounds among others and the use of microbial fuel cells are finally discussed.

Decontamination of wastewaters containing synthetic organic dyes by electrochemical methods. An updated review

Removal of synthetic dyes from wastewaters: a review.

Nanocrystal quantum dots (QDs) have been applied to molecular biology because of their greater and longer fluorescence. Here we report the potential cytotoxicity of our characterized QDs modified with various molecules. Surface modification of QDs changed their physicochemical properties. In addition, the cytotoxicity of QDs was dependent on their surface molecules. These results suggested that the properties of QDs are not related to those of QD-core materials but to molecules covering the surface of QDs.

Physicochemical Properties and Cellular Toxicity of Nanocrystal Quantum Dots Depend on Their Surface Modification

We determined the genotoxicity of 39 chemicals currently in use as food additives. They fell into six categories—dyes, color fixatives and preservatives, preservatives, antioxidants, fungicides, and sweeteners. We tested groups of four male ddY mice once orally with each additive at up to 0.5×LD50 or the limit dose (2000 mg/kg) and performed the comet assay on the glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow 3 and 24 h after treatment. Of all the additives, dyes were the most genotoxic. Amaranth, Allura Red, New Coccine, Tartrazine, Erythrosine, Phloxine, and Rose Bengal induced dose-related DNA damage in the glandular stomach, colon, and/or urinary bladder. All seven dyes induced DNA damage in the gastrointestinal organs at a low dose (10 or 100 mg/kg). Among them, Amaranth, Allura Red, New Coccine, and Tartrazine induced DNA damage in the colon at close to the acceptable daily intakes (ADIs). Two antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)), three fungicides (biphenyl, sodium o-phenylphenol, and thiabendazole), and four sweeteners (sodium cyclamate, saccharin, sodium saccharin, and sucralose) also induced DNA damage in gastrointestinal organs. Based on these results, we believe that more extensive assessment of food additives in current use is warranted.

The comet assay with 8 mouse organs: results with 39 currently used food additives.

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology.Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced b...

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database.

DNA damage induced by red food dyes orally administered to pregnant and male mice.

The effect of 2 model chemical mutagens on DNA was evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in 5 mouse organs--liver, lung, kidney, spleen and bone marrow. Mice were sacrificed 3 and 24 h after the administration of the direct mutagen ethyl nitrosourea (ENU) or the liver-targeting promutagen p-dimethylaminoazobenzene (DAB). Each organ was minced, suspended at a concentration of 1 g/ml in chilled homogenizing buffer (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, homogenized gently using a Potter-type homogenizer at 500-800 rpm set in ice, and then centrifuged nuclei were used for the alkaline SCG assay. ENU induced DNA damage in cells all of the organs studied DAB, on the other hand, produced a positive response in the liver only. We suggest that it may be possible to use the alkaline SCG assay using a homogenization technique to detect the genotoxicity of chemicals in vivo in their target organs.

Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single cell gel electrophoresis (Comet) assay.

Yu F. Sasaki

Papers

Physicochemical Properties and Cellular Toxicity of Nanocrystal Quantum Dots Depend on Their Surface Modification

The comet assay with 8 mouse organs: results with 39 currently used food additives.

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database.

DNA damage induced by red food dyes orally administered to pregnant and male mice.

Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single cell gel electrophoresis (Comet) assay.