ABSTRACT
Two modifications of the rinsing procedure within the BCOP assay were proposed. Their ability to enhance the removal efficiency of highly viscous and colored samples was compared with an unmodified BCOP procedure (TG OECD 437). The first modification consisted of three-step washing of the applied chemicals from the cornea using Eagle's Minimum Essential Medium (EMEM), olive oil and EMEM, while the classical OECD TG 437 procedure prescribes only EMEM. Within the second modification, mechanical removal of the tested sample from the cornea surface prior to the two step washing procedure was performed. The in vitro irritation score (IVIS) exceeded the value of 55 for 9 out of 20 samples when a non-modified rinsing procedure was used. The first modification with the olive oil resulted in a decrease in IVIS for numerous samples, while an IVIS score drop below the threshold value of 55 was only observed for two of them. Mechanical removal of sample residua resulted in a further decline in the measured IVIS. Only the three samples treated by means of this procedure revealed an IVIS above 55. The decreases in IVIS observed during both modifications were mainly related to the reduced opacity, whereas the permeability mostly remained unaffected.
Subject(s)
Biological Assay/methods , Cornea/drug effects , Corneal Opacity , Irritants/toxicity , Animal Testing Alternatives , Animals , Cattle , Color , Cornea/metabolism , In Vitro Techniques , Olive Oil , Permeability , ViscosityABSTRACT
Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.
Subject(s)
Chromatography, High Pressure Liquid/methods , Histone Acetyltransferases/genetics , Polymorphism, Genetic , Trans-Activators/genetics , Trinucleotide Repeats , Base Sequence , Breast Neoplasms/genetics , DNA Primers/genetics , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Female , Humans , Nuclear Receptor Coactivator 3 , Nucleic Acid DenaturationABSTRACT
Complete or partial inability to sense and repair DNA damage increases the risk of developing cancer. The ataxia telangiectasia mutated (ATM) protein kinase has a crucial role in response to DNA double-strand breaks. Hereditary mutations in the ATM gene are the cause of a rare genomic instability syndrome ataxia telangiectasia (AT) characterized, among others, by elevated cancer risk. Although clear in homozygotes, numerous studies have failed to find a link between heterozygotes and cancer. However, there is increasing evidence that ATM heterozygotes have an increased risk of developing breast cancer. First, epidemiological studies conferred an increased risk of breast cancer among AT relatives. Second, in vitro studies of heterozygous cells provide strong evidence of hyperradiosensitivity. Third, some clinical studies found an increased frequency of ATM mutations among high-risk breast cancer families.
