Two thousand cells were counted and values of apoptotic and necrotic cells are given as percentages. For the determination of chromosomal aberrations, we added 0.4 μg/mL colcemide to the dishes (triplicate/animal/NDEA group concentration) and incubated the cells for a further 3 h. The medium was replaced by 2 mL collagenase solution (0.5 mg/mL) and incubated for 10 min in order to detach the cells. The cells were collected by centrifugation, and 0.01 M KCl hypotonic solution was added for 10 min. The cells were fixed overnight in cold methanol–glacial acetic acid (3:1) by dropping the suspension
on glass slides. Five slides were prepared for each animal and NDEA check details group concentration. The slides were stained for 15 min using 4.5 μg/mL Hoechst 33258, rinsed with distilled water, mounted in PBS, pH 7.0, with a coverglass, and exposed to a 40 W blacklight lamp (Philips TLD 36W08) at 50 °C for 1 h. After removing the
coverslips, the slides were stained with 5% Giemsa solution. Chromosomal aberrations were scored using 50 well-spread metaphases, and aberration numbers were given per diploid cell, i.e. 42 chromosomes. Metaphases were scored for chromosome-type aberrations, such as chromosome deletions, dicentrics and ring chromosomes. The data were analyzed by one-way analysis of variance (ANOVA) and the results were considered statistically significant at p < 0.05. Total RNA was isolated with TRIzol (Invitrogen, Germany) buy Stem Cell Compound Library and treated with 0.5 units DNase I (Invitrogen) according to the manufacturer’s instructions. DNA-free RNA was dissolved in DEPC water and stored at −80 °C. First-strand L-gulonolactone oxidase cDNA synthesis was performed using an oligo dT primer (0.3 ng/μL) and Superscript™ III bulk mix (Invitrogen) according to the manufacturer’s instructions. The DNA coding sequences of CYP genes were obtained
from GenBank (http://www.ncbi.nml.nih.gov/GenBank), and the entry codes are given in Table 1. A subfamily-specific DNA region was selected as the site of hybridization for each CYP for the design of either the forward or the reverse primer. The corresponding oligonucleotide was selected for amplification on the basis of (i) similar melting temperatures, (ii) similar nucleotide length, and (iii) generation of an amplicon with at least 50% GC content. To control specificity, all the primers were submitted to the basic logarithmic alignment search tool (BLAST). Quantitative RT PCR was done using the BioRad iCycler iQ Real-Time Detection System (BioRad) according to the manufacturer’s instructions. A cycle threshold (CT) is defined as the cycle number at which the fluorescence generated within a reaction is significantly higher than the background value, and is inversely proportional to the relative expression level of a gene.