Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup.

The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93).

Detection of Pig-a Mutant Erythrocytes in the Peripheral Blood of Rats and Mice.

The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because: (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection.

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints.

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays.

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset.

An ultra low-cost smartphone device for in-situ monitoring of acute organophosphorus poisoning for agricultural workers.

In this work, we present an ultra-low-cost smartphone device for in situ quantification of OP poisoning severity. The performance of the lens-less smartphone spectrum apparatus (LeSSA) is evaluated using standard human Interleukin-6 (IL-6) immunoassay kits. Upon dose-response curve fitting, LeSSA demonstrates an accuracy of 99.

Suitability of Long-Term Frozen Rat Blood Samples for the Interrogation of Pig-a Gene Mutation by Flow Cytometry.

The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection.

Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine.

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC.

In Vivo Pig-a gene mutation assay: Guidance for 3Rs-friendly implementation.

The rodent Pig-a assay is an in vivo method for the detection of gene mutation, where lack of glycosylphosphatidylinositol-anchored proteins on the surface of circulating red blood cells (RBCs) serves as a reporter for Pig-a gene mutation. In the case of rats, the frequency of mutant phenotype RBCs is measured via fluorescent anti-CD59 antibodies and flow cytometry. The Pig-a assay meets the growing expectations for novel approaches in animal experimentation not only focusing on the scientific value of the assay but also on animal welfare aspects (3Rs principles), for example, amenable to integration into pivotal rodent 28-day general toxicology studies.

Detection of Pig-a mutant erythrocytes in the peripheral blood of rats and mice.

The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection.

Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse.

Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed.

Mutant frequency in comparison to oxidative DNA damage induced by ochratoxin A in L5178Y tk+/- (3.7.2C) mouse lymphoma cells.

Ochratoxin A (OTA) is a naturally occurring mycotoxin that contaminates animal feed and human food. OTA is nephrotoxic, hepatotoxic, immunosuppressive and a potent renal carcinogen in rodents. In the present study, we evaluated the genotoxicity of OTA in L5178Y tk(+/-) (3.

Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens.

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g.

Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, aristolochic acids.

Aristolochic acids (AAs) are carcinogenic plant toxins that are relatively strong gene mutagens, both in vitro and in vivo, but weak inducers of micronuclei in vivo. In order to clarify the reasons for these disparate responses, we evaluated the genotoxicity of AAs in F344 rats using several assays that respond to DNA damage in bone marrow. Groups of 7- to 8-week-old male rats (n=6) were gavaged with 0, 2.

Genotoxicity of TiO(2) anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays.

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO(2)-NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.

Manifestation and persistence of Pig-a mutant red blood cells in C57BL/6 mice following single and split doses of N-ethyl-N-nitrosourea.

Treating rats with single doses of N-ethyl-N-nitrosourea (ENU) results in a time-dependent accumulation of Pig-a-mutant phenotype peripheral red blood cells (RBCs), reaching a plateau at about 6-weeks posttreatment, with the response persisting for at least 26 weeks. In the present study, groups of 5 C57BL/6 male mice were administered single i.p.

Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea-comparison with other in vivo genotoxicity endpoints.

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon.

Report on stage III Pig-a mutation assays using benzo[a]pyrene.

Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S.

International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories.

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3).

Comparative analysis of micronuclei and DNA damage induced by Ochratoxin A in two mammalian cell lines.

The fungal toxin, Ochratoxin A (OTA), is a common contaminant in human food and animal feed. The present study evaluated micronucleus (MN) induction by OTA in comparison with its ability to induce cytotoxicity and DNA damage in two mammalian cell lines, CHO-K1-BH(4) Chinese hamster ovary cells and TK6 human lymphoblastoid cells. Micronuclei were evaluated by flow cytometry, cytotoxicity was estimated by relative population doubling (RPD), while direct DNA damage and oxidative DNA damage were measured with the Comet assay, performed without and with digestion by formamidopyrimidine-DNA glycosylase (fpg).

Genotoxicity of heptachlor and heptachlor epoxide in human TK6 lymphoblastoid cells.

The genotoxic potential of the organochlorine insecticides heptachlor (HC) and its metabolite heptachlor epoxide (HCE) has been evaluated in TK6 cells, a well-established human lymphoblastoid cell line. Genotoxicity has been determined by scoring the induction of DNA breaks in the comet assay and by measuring the frequency of micronuclei (MN) in binucleated cells. The results indicate that both compounds are able to induce significant increases in the percentage of DNA in the tail, the parameter used in the comet assay, with a direct dose-response relationship.

DNA damage in Pakistani agricultural workers exposed to mixture of pesticides.

A cross-sectional study was designed to determine whether occupational exposure to a complex mixture of pesticides results in a significant increase of DNA damage in farmers chronically exposed to pesticides in open fields. Leukocytes from 47 agriculture workers exposed to pesticides and 50 controls were evaluated with comet assay. Workers recruitment was based on their exposure to pesticides during the spraying season on cotton crop.

Cytogenetic damage in female Pakistani agricultural workers exposed to pesticides.

Bhawalpur is a major cotton-growing area in Pakistan. Cotton picking in Pakistan is carried out by females and as a result of the intensive use of pesticides during the growing season these females are exposed to pesticide residues in the picking season. In the present study, peripheral blood was obtained from 69 cotton pickers and 69 unexposed females and used to assess the effect of pesticide exposure on genetic damage as well as on hepatic enzymes and serum cholinesterase.

DNA damage in Pakistani pesticide-manufacturing workers assayed using the Comet assay.

The production and use of chemical pesticides has increased in recent years. Although the increased use of pesticides may benefit agriculture, they are also the potential source of environmental pollution, and exposure to pesticides can have negative consequences for human health. In the present study, we have assessed DNA damage in blood leukocytes from 29 Pakistani pesticide-factory workers and 35 controls of similar age and smoking history.

Cytogenetic analysis of Pakistani individuals occupationally exposed to pesticides in a pesticide production industry.

Although several cytogenetic biomonitoring studies on workers exposed to pesticides have been reported, there is only limited information on this topic from developing countries where pesticides have been widely used over the years. People in developing countries are at higher risk from exposure, due to poor working conditions and a lack of awareness of the potential hazards during manufacturing and application of the pesticides. The present study has assessed the genotoxic effects of pesticides on workers involved in the pesticide manufacturing industry.