Human TK6 cells treated with BPDE or N-OH-AABP yielding 5%, 15% and 40% of toxicity

Cell culture Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring. Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere. Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL. Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate. Chemical treatment, mutagenesis and survival measurements N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%. Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”. Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction. At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC. DNA microarrays Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs. The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases. For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA). Each clone was represented once per array. Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5 RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization. The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software. Keywords: repeat sample