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Publications (87)
A multi-sector study (i.e., Ring Trial) was designed to improve the in vitro detection of N-nitrosamine (NA)-associated mutagenicity by optimizing the bacterial reverse mutation (i.e., Ames) assay protocol and testing various conditions on the sensitivity and specificity for the prediction of rodent carcinogenicity. A total of 29 NAs and 3 N-nitros...
The current genotoxicity testing paradigm provides little mechanistic information, has poor specificity in predicting carcinogenicity in humans, and is not suited to assessing a large number of chemicals. Genomic technologies enable the characterization of genome‐wide transcriptional changes in response to chemical treatments that can inform mechan...
Gene expression biomarkers have the potential to identify genotoxic and non‐genotoxic carcinogens, providing opportunities for integrated testing and reducing animal use. In August 2022, an International Workshops on Genotoxicity Testing (IWGT) workshop was held to critically review current methods to identify genotoxicants using transcriptomic pro...
As a part of the International Workshop on Genotoxicity Testing (IWGT) in 2022, a workgroup was formed to evaluate the level of validation and regulatory acceptance of transcriptomic biomarkers that identify genotoxic substances. Several such biomarkers have been developed using various molecular techniques and computational approaches. Within the...
The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal So...
Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points‐of‐departures (PODs) f...
Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departure (PODs) for se...
Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mech...
There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets o...
The unexpected detection of nitrosamine impurities in human medicines has recently seen global regulators act to understand the risks of these contaminations to patients and to limit their presence. Over 300 nitrosamines are known, many of which are highly potent mutagenic carcinogens. Regulators first became aware of the presence of nitrosamines i...
We present a hypothetical case study to examine the use of a next‐generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmac...
Background:
N-nitrosodimethylamine (NDMA) has been classified as a probable human carcinogen and has been found as a contaminant in valsartan, an antihypertensive drug. Potential carcinogenic effects from the consumption of NDMA-contaminated valsartan have not been analyzed to date in large-scale cohort studies. We therefore carried out the study...
MicroRNAs (miRNAs) are small non-coding RNA that regulate the expression of messenger RNA and are implicated in almost all cellular processes. Importantly, miRNAs can be released extracellularly and are stable in these matrices where they may serve as indicators of organ or cell-specific toxicity, disease, and biological status. There has thus been...
With sound understanding of biological concepts, the notion of threshold effect levels has grown in acceptance especially for electrophile-induced mutations. However, mutagenesis is only one part of the exposure-to-tumor process in chemical carcinogenesis. Another important part is carcinogen-induced cell death and senescence that eliminates or imm...
Robust genomic approaches are now available to realize improvements in efficiencies and translational relevance of cancer risk assessments for drugs and chemicals. Mechanistic and pathway data generated via genomics provide opportunities to advance beyond historical reliance on apical endpoints of uncertain human relevance. Published research and r...
Genotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world-wide for analysis of drug candidates, and environmental and industrial chemicals. Current genotoxicity testing batteries feature a high incidence of irrelevant positive findings—particularly for in vitro chromosomal damage (CD) ass...
In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs...
An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the ob...
As part of the 7th International Workshops on Genotoxicity Testing held in Tokyo, Japan in November 2017, a workgroup of experts reviewed and assessed the risk of aneugens for human health. The present manuscript is one of three manuscripts from the workgroup and reports on the unanimous consensus reached on the evidence for aneugens affecting germ...
Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical aneugens; and chemotherapy-related malignant neop...
Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo tes...
Background
Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM...
The major obstacle in the clinical use of the antitumor drug cisplatin is inherent and acquired resistance. Typically, cisplatin resistance is not restricted to a single mechanism demanding for a systems pharmacology approach to understand a whole cell’s reaction to the drug. In this study, the cellular transcriptome of untreated and cisplatin-trea...
Significance
Standard in vitro assays to assess genotoxicity frequently generate positive results that are subsequently found to be irrelevant for in vivo carcinogenesis and human cancer risk assessment. Currently used follow-up methods, such as animal testing, are expensive and time-consuming, and the development of approaches enabling more accura...
Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts no...
The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDD...
Analysis of pAtm.
Western Blot Analysis of pAtm (n = 3) a) as integrated signal intensity normalized to the housekeeper α-actin in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Western blot pATM.
Western Blot of pATM in A549 and A549rCDDP2000 cells. Lanes 1, 6, 11,: A549 untreated; lanes 2, 7, 12, A549 cells treated with 11 μM cisplatin; lanes 3, 8, 13, A549rCDDP2000 untreated; lanes 4, 9, 14, A549rCDDP2000 treated with 11 μM cisplatin; lanes 5, 10, 15, A549rCDDP2000 treated with 34 μM cisplatin.
(TIF)
Western blot MDM2.
Western Blot of MDM2 in A549 (caption in black letters) and A549rCDDP2000 (caption in red letters) cells showing lanes of A549 untreated (caption ctrl), A549 treated with 11 μM cisplatin (caption 11), A549rCDDP2000 untreated (caption ctrl), A549rCDDP2000 treated with 11 μM cisplatin (caption 11) and A549rCDDP2000 treated with 34...
Western blot SIP.
Western Blot of SIP in A549 (caption in black letters) and A549rCDDP2000 (caption in red letters) cells showing lanes of A549 untreated (caption ctrl), A549 treated with 11 μM cisplatin (caption 11), A549rCDDP2000 untreated (caption ctrl), A549rCDDP2000 treated with 11 μM cisplatin (caption 11) and A549rCDDP2000 treated with 34 μM...
Western blot GADD45a.
Western Blot of GADD45a in A549 (caption in black letters) and A549rCDDP2000 (caption in red letters) cells showing lanes of A549 untreated (caption ctrl), A549 treated with 11 μM cisplatin (caption 11), A549rCDDP2000 untreated (caption ctrl), A549rCDDP2000 treated with 11 μM cisplatin (caption 11) and A549rCDDP2000 treated wi...
Analysis of p21.
Analysis of p21 in a) RT-PCR (n = 3) as individual data points presented as mean ± SEM and b) Western Blot (n = 3) as integrated signal intensity normalized to the housekeeper α-actin in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Analysis of SIP.
Analysis of SIP in a) RT-PCR (n = 3) as individual data points presented as mean ± SEM and b) Western Blot (n = 3) as integrated signal intensity normalized to the housekeeper GAPDH in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Analysis of XPC.
Analysis of XPC in a) RT-PCR (n = 3) as individual data points presented as mean ± SEM and b) Western Blot (n = 3) as integrated signal intensity normalized to the housekeeper GAPDH in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Analysis of GADD45a.
Analysis of GADD45a in a) RT-PCR (n = 3) as individual data points presented as mean ± SEM and b) Western Blot (n = 3) as integrated signal intensity normalized to the housekeeper GAPDH in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Western blot p53.
Western Blot of p53 in A549 and A549rCDDP2000 cells. Lanes 1, 6, 11,: A549 untreated; lanes 2, 7, 12, A549 cells treated with 11 μM cisplatin; lanes 3, 8, 13, A549rCDDP2000 untreated; lanes 4, 9, 14, A549rCDDP2000 treated with 11 μM cisplatin; lanes 5, 10, 15, A549rCDDP2000 treated with 34 μM cisplatin.
(TIF)
Western blot p21.
Western Blot of p21 in A549 and A549rCDDP2000 cells. Lanes 1, 6, 11,: A549 untreated; lanes 2, 7, 12, A549 cells treated with 11 μM cisplatin; lanes 3, 8, 13, A549rCDDP2000 untreated; lanes 4, 9, 14, A549rCDDP2000 treated with 11 μM cisplatin; lanes 5, 10, 15, A549rCDDP2000 treated with 34 μM cisplatin.
(TIF)
Analysis of MDM2.
Analysis of MDM2 in a) RT-PCR (n = 3) as individual data points presented as mean ± SEM and b) Western Blot (n = 3) as integrated signal intensity normalized to the housekeeper GAPDH in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Western blot XPC.
Western Blot of XPC in A549 (caption in black letters) and A549rCDDP2000 (caption in red letters) cells showing lanes of A549 untreated (caption ctrl), A549 treated with 11 μM cisplatin (caption 11), A549rCDDP2000 untreated (caption ctrl), A549rCDDP2000 treated with 11 μM cisplatin (caption 11) and A549rCDDP2000 treated with 34 μM...
Western blot of GAPDH for GADD45a normalization.
Western Blot of GAPDH for GADD45a normalization in A549 (caption in black letters) and A549rCDDP2000 (caption in red letters) cells showing lanes of A549 untreated (caption ctrl), A549 treated with 11 μM cisplatin (caption 11), A549rCDDP2000 untreated (caption ctrl), A549rCDDP2000 treated with 11 μM...
Analysis of p53.
Analysis of p53 in a) RT-PCR (n = 3) as individual data points presented as mean ± SEM and b) Western Blot (n = 3) as integrated signal intensity normalized to the housekeeper α-actin in A549 and A549rCDDP2000 cells, presented as mean ± SEM.
(TIF)
Tables with individual data values.
Tables with individual data values analysed for preparation of all figures in this manuscript.
(DOCX)
Western Blot of α-Actin for p53 pATM and p21 normalization.
Western Blot of α-Actin in A549 and A549rCDDP2000 cells. Lanes 1, 6, 11,: A549 untreated; lanes 2, 7, 12, A549 cells treated with 11 μM cisplatin; lanes 3, 8, 13, A549rCDDP2000 untreated; lanes 4, 9, 14, A549rCDDP2000 treated with 11 μM cisplatin; lanes 5, 10, 15, A549rCDDP2000 treated wit...
Objective: Adjuvant GBM chemotherapy is carried out with the alkylating agent Temozolomide (TMZ). The aggressiveness of GBM is mainly based on resistance to radio- and chemotherapy. In order to understand the underlying molecular mechanisms of resistance we analysed the response of the intrinsic resistant GBM cell line U251 to TMZ. Following in vit...
This guide describes all practical aspects of genetic toxicology testing of chemicals and other materials in a GLP environment; it covers those tests normally used for screening and regulatory submissions. Method setup and validation, study design, test performance, collection and evaluation of results as well as reporting are described in unparall...
Download free full text here:
http://authors.elsevier.com/a/1Se1O~81I44Ki
Narrow Therapeutic Index Drugs (NTIDs) are characterized by a small range between therapeutic and toxicological effect. Missing international harmonized definition for NTIDs the EMA does not even have a definition of NTIDs in contrast to the U.S. FDA, Health Canada, and the...
The in vivo comet assay is an extremely fast, flexible, and sensitive assay adaptable to a variety of exposure conditions and applicable to almost any tissue or cell type. This makes it an ideal option for the safety testing of new or previously tested products that require quick results, specific exposure conditions, and/or target organ assessment...
The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options...
This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose-response analysis of genetic toxicology data,...
This report summarizes the discussion, conclusions, and points of consensus of the IWGT Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (QWG) based on a meeting in Foz do Iguaçu, Brazil October 31-November 2, 2013. Topics addressed included (1) the need for quantitative dose-response analysis, (2) methods to analyze e...
The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay. Altho...
A generally applicable highly effective purification method for antiviral plant proteins, selective for those with ribosome-inactivating properties, was developed. It involved affinity chromatography on Cibacron Blue Sepharose of the extracted plant sap after acid-ethanol precipitation and finally cation exchange chromatography on Mono S (Pharmacia...
Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Geno...
This chapter describes the current status and impact of data from microarray applications on drug development and approval from a European regulator’s point of view.
Microarray technology is regarded as a new, valuable and increasingly important tool in drug development and risk evaluation. The technique may enable faster development of new and saf...
Damage to DNA can trigger a variety of stress-related signals that alter the expression of genes associated with numerous biological pathways. In this study, we have used a cDNA microarray representing 1089 genes related to DNA damage and repair, cell cycle, transcription, metabolism and other toxicologically important cell functions to identify ge...
The frequency of functionally important mutations and alleles of genes coding for xenobiotic metabolizing enzymes shows a wide ethnic variation. However, little is known of the frequency distribution of the major allelic variants in the Russian population.
Using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) genotypin...
Molecular genetic parameters are not only important in medical diagnostics but they are also the basic element of pharmacogenetics and of great importance for a personalized drug therapy. The development of an individualized therapeutic strategy for each patient will become an exciting challenge. Genetic polymorphisms are responsible for distinct i...
Enzyme-specific testing for drug interactions by in vitro techniques has become a routine practice in drug development. With many drugs, enzyme induction has similar importance for the prediction of drug-drug interactions. We developed a method for recognizing enzyme induction mediated via the aryl hydrocarbon receptor. This type of induction may b...
The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively...
Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet...
