ERCC1: a comparative genomic perspective.

ERCC1 plays an essential role in the nucleotide excision repair (NER) of DNA. We compare 37 kb of sequence from the ERCC1 region on human chromosome 19q13.3 to the orthologous region on mouse chromosome 7. In addition to showing the conserved gene structure between ERCC1, ASE-1, and their murine counterparts, this genomic comparison reveals a highly conserved 497 bp segment found 5 kb upstream of ERCC1 exon 1 that contains a CpG island and previously unidentified "classical" promoter elements. Additional putative regulatory elements are also found within a conserved LINE-1 (long interspersed nuclear element) sequence 800 bp upstream of exon 1 in both human and mouse. Expressed sequence tag (EST) assemblies for human ERCC1 identified numerous splice variants involving exons 1, 2, 3, 7, 8, and 9 that could affect DNA repair efficiencies of ERCC1. A previously undescribed transcript that reads through exon 9 and utilizes the polyadenylation signal of a neighboring Alu element accounts for nearly half of the total splice variants identified in the human EST database. This transcript would theoretically translate to a larger ERCC1 protein product containing a novel C-terminal end. Overall, approximately 18% of publicly available ERCC1 cDNA sequences were determined to be splice variants, while no variants were found in the mouse. The ability to assess novel transcripts and identify candidate regulatory regions demonstrates the potential utility for a catalogue archiving comparative analyses for all genes involved in DNA repair. Our comparative genomic analysis of ERCC1 can be viewed at http://web.uvic.ca/-bioweb/laj.html.

Three-dimensional transgenic cell model to quantify genotoxic effects of space environment.

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of multiple copies of defined target genes for genotoxic assessment. Rat 2 lambda fibroblasts, genetically engineered to contain high-density target genes for mutagenesis (Stratagene, Inc., Austin, TX), were cocultured with human epithelial cells on Cytodex beads in the High Aspect Ratio Bioreactor (Synthecon, Inc, Houston, TX). Multi-bead aggregates were formed by day 5 following the complete covering of the beads by fibroblasts. Cellular retraction occurred 8-14 days after coculture initiation culminating in spheroids retaining few or no beads. Analysis of the resulting tissue assemblies revealed: multicellular spheroids, fibroblasts synthesized collagen, and cell viability was retained for the 30-day test period after removal from the bioreactor. Quantification of mutation at the LacI gene in Rat 2 lambda fibroblasts in spheroids exposed to 0-2 Gy neon using the Big Blue color assay (Stratagene, Inc.), revealed a linear dose-response for mutation induction. Limited sequencing analysis of mutant clones from 0.25 or 1 Gy exposures revealed a higher frequency of deletions and multiple base sequencing changes with increasing dose. These results suggest that the three-dimensional, multicellular tissue assembly model produced in NASA bioreactors are applicable to a wide variety of studies involving the quantification and identification of genotoxicity including measurement of the inherent damage incurred in Space.

Msh2 DNA mismatch repair gene deficiency and the food-borne mutagen 2-amino-1-methy1-6-phenolimidazo [4,5-b] pyridine (PhIP) synergistically affect mutagenesis in mouse colon.

Msh2 deficiency and food-borne carcinogen PhIP have been implicated as genetic and environmental factors, respectively, in human colon carcinogenesis. It is not clear whether loss of one or both alleles of Msh2 gene increases the mutational sensitivity in colon when exposed to environmental carcinogens. In the current study, Msh2(+/-)/lacI and Msh2(-/-)/lacI double transgenic mice were treated with PhIP and mutations in the lacI gene were studied in the colon. The spontaneous mutation frequency (MF) is approximately eightfold higher in Msh2(-/-) mice than in Msh2(+/+) mice, while Msh2(+/-) mice display similar levels of spontaneous mutation as the Msh2 wild type mice. PhIP induced a significant increase in MF in all genotypes of mice. However, induced MF is much higher in Msh2(-/-) mice compared to Msh2(+/+) and Msh2(+/-) mice. Msh2(+/-) mice displayed an increased level of G:C>T:A transversions and -1 frameshifts upon PhIP treatment. In contrast, loss of both Msh2 alleles mainly results in increased frequency of G:C>A:T transitions when exposed to PhIP. These results suggest that a defect in mismatch repair may result in an enhanced sensitivity from exposure to a dietary carcinogen. It also provides insight into interaction between genetic and environmental factors in human carcinogenesis.

Protective effect of green tea against benzo[a]pyrene-induced mutations in the liver of Big Blue transgenic mice.

We assessed the ability of green tea to protect against benzo[a]pyrene (B[a]P)-induced mutations in the liver of lacI transgenic male C57BL/6 Big Blue mice. The mice were given a 2% Japanese green tea hot water extract as their sole source of drinking water for 10 weeks. After 7 weeks, they received a total dose of 150 mg/kg B[a]P. Treatment with B[a]P resulted in a two-fold higher lacI mutant frequency than the untreated controls (8.6+/-0.8 x 10(-5) versus 4.0+/-0.7 x 10(-5), P=0.01). B[a]P increased the frequency of its characteristic mutation (GC-->TA transversions) nearly five-fold, from 0.75 x 10(-5) to 3.7 x 10(-5). In mice treated with green tea, the induced B[a]P mutant frequency decreased by 63%, while GC-->TA transversions were reduced by 54%. Thus, we report evidence that green tea extract significantly suppressed B[a]P-induced mutation by lowering its specific transversion mutation in the lacI transgene in vivo. Further studies will address the correlation between the modulation of metabolic enzymes and the protection against induced mutation by green tea.

Mutations induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats. In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats. This is the first report of mutational data from discrete regions of the colon. After 61 days of treatment with 200 p.p.m. PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls. PhIP-induced mutant frequencies increased significantly (overall trend, P < 10(-4)) along the length of the colon of both males and females, with cecum < proximal colon < distal colon. A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females. These mutational spectra consisted predominantly of G:C-->T:A and G:C-->C:G transversions and deletions of G:C base pairs. There were no significant differences between the mutational spectra with respect to sex or position in the colon. Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats.

Mutagenicity of TCDD in Big Blue transgenic rats.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a significant environmental contaminant resulting from such industrial processes as pulp and paper production. TCDD is a suspected human carcinogen and its ability to induce cancer in laboratory rodents is well documented. Its mechanism of tumor initiation, however, is not well understood and in vitro mutagenicity studies have yielded inconsistent results. In this study, Big Blue lacI transgenic rats were used to assess the mutagenicity of TCDD in both male and female animals. After 6 weeks of exposure to 2 microg/kg TCDD neither an increase in mutation frequency nor any change in mutation spectrum was observed in either male or female animals.

Radiation induced apoptosis in ataxia telangiectasia homozygote, heterozygote and normal cells.

Recent reports suggest that the radiation-induced, p53-dependent, apoptotic response is aberrant in ataxia telangiectasia (AT) cells. We investigated the possibility that an aberrant apoptotic response to ionizing radiation may also be the characteristic of AT heterozygotes and may facilitate in discriminating AT heterozygotes from the general population. Log phase, Epstein Barr virus (EBV) transformed lymphoblastoid cell lines and primary lymphocytes from three AT families were irradiated and the apoptotic response at 30h post radiation was measured by flow cytometry using TUNEL and hypodiploid methods. Our results show that the apoptotic response of AT homozygote (ATM-/-), AT heterozygote (ATM+/-) and normal cells (ATM+/+) to ionizing radiation, measured by the hypodiploid and TUNEL methods using flow cytometry, is dose and time dependent. Furthermore, this response is paradoxical in that ATM (-/-) lymphoblastoid cells were characterized by a reduced post radiation apoptotic response compared to their normal counterparts. Heterozygote (ATM+/-) lymphoblastoid cells displayed an intermediate response to ionizing radiation. In contrast, primary, non-transformed AT cells exhibited the same apoptotic response as their normal counterparts. Our results thus indicate that pre-radiation, EBV-transformed, lymphoblastoid cell lines from individual families may be useful in discriminating ATM status, but patient-derived, primary AT homozygous, heterozygous and normal primary cultured lymphocytes cannot be discriminated by this assay.

Human DNA repair genes.

DNA repair systems are essential for the maintenance of genome integrity. Consequently, the disregulation of repair genes can be expected to be associated with significant, detrimental health effects, which can include an increased prevalence of birth defects, an enhancement of cancer risk, and an accelerated rate of aging. Although original insights into DNA repair and the genes responsible were largely derived from studies in bacteria and yeast, well over 125 genes directly involved in DNA repair have now been identified in humans, and their cDNA sequence established. These genes function in a diverse set of pathways that involve the recognition and removal of DNA lesions, tolerance to DNA damage, and protection from errors of incorporation made during DNA replication or DNA repair. Additional genes indirectly affect DNA repair, by regulating the cell cycle, ostensibly to provide an opportunity for repair or to direct the cell to apoptosis. For about 70 of the DNA repair genes listed in Table I, both the genomic DNA sequence and the cDNA sequence and chromosomal location have been elucidated. In 45 cases single-nucleotide polymorphisms have been identified and, in some cases, genetic variants have been associated with specific disorders. With the accelerating rate of gene discovery, the number of identified DNA repair genes and sequence variants is quickly rising. This report tabulates the current status of what is known about these genes. The report is limited to genes whose function is directly related to DNA repair.

Alterations of K-ras and p53 mutations in colorectal cancer patients in Central Europe.

Many molecular investigations of colorectal cancer (CRC) have suggested that the accumulation of specific mutations in proto-oncogenes and tumor suppressor genes regulating cell growth via signal transduction trigger the stagewise progression to malignancy. In this study, the frequency, location, and type of mutations of the K-ras proto-oncogene exon I and p53 tumor suppressor gene exons 5-8 were analyzed in colorectal carcinomas of 65 patients from Central Europe, using polymerase chain reaction (PCR)-cold single-strand conformation polymorphism (SSCP) screening and direct sequencing. The incidence of K-ras activating mutations in these Central European samples was lower (25%) compared to that obtained in American and western European populations (40-50% at least), while the incidence of p53 inactivating mutations was similar (58%). These results suggest that some other genetically linked mechanisms may play a role in CRC development and progression, and hence K-ras and p53 mutations cannot be considered to be universal genetic markers for CRC.

Spontaneous mutation of the lacI transgene in rodents: absence of species, strain, and insertion-site influence.

Comparison of spontaneous mutation spectra derived from different transgenic constructs can provide valuable insights for interpreting the mechanisms of spontaneous mutation. In this study, spontaneous mutation frequencies and spectra of the lacI transgene are compared in the liver of C57BL/6, B6C3F1, and BC-1 mice and F344 rats. Before correction for clonal expansion, the mutant frequency varied from 2.6 +/- 0.45 to 5.0 +/- 2.4 x 10(-5). Correction for potential clonal expansion reduced the range in mutation frequency to between 2.3 +/- 0.45 and 3.5 +/- 2.0 x 10(-5). There is thus no statistical difference in spontaneous mutation frequency between the different strains and species. G:C --> A:T transitions and to a lesser extent, G:C --> T:A transversions dominate the mutational spectra in all of these animals. In three strains of mice, G:C --> A:T transitions account for 50.7-53.3% of mutation, 81.7-83.8% of which involve CpG sites, whereas G:C --> T:A transversions account for 17.8-32.9% of mutations with 43.2-50.0% found at CpG sites. In rats, G:C --> A:T transitions account for 38.0% of the spectra, 70.0% of which involve CpG sites, whereas G:C --> T:A transversions account for 23.0% of the spectra, 70.0% of which involve CpG sites. The distribution of other classes of mutations is also very similar. We conclude that, despite reports about species and strain differences in induced mutation, spontaneous mutations in the lacI transgene appear to be similar, regardless of genomic location, rodent strain, or species. In addition to insights into spontaneous mutation, this study also provides essential data for comparison with and interpretation of induced mutations.

Recent advances in research on radiofrequency fields and health.

Since the Royal Society of Canada report on potential health risks of radiofrequency (RF) fields from wireless telecommunications in the spring of 1999, there have been several newly published reports on risks associated with the use of mobile phones. This article provides a summary of scientific research on the potential health effects of radiofrequency fields that has been reported since the original Royal Society report was published. This update also discusses several earlier results not included in the original report.

Modulation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mutation in the cecum and colon of big blue rats by conjugated linoleic acid and 1,2-dithiole-3-thione.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent mutagen and suspected human carcinogen present in cooked protein-rich food. It preferentially induced colon tumors in male rats and mammary tumors in female rats. In the present study, the in vivo antimutagenic efficacy of two dietary compounds, conjugated linoleic acid (CLA) and 1,2-dithiole-3-thione (DTT), against PhIP was explored using 1acI transgenic Big Blue rats. Five- or six-week-old male Big Blue rats were fed a diet containing CLA (0.5%, wt/wt) or DTT (0.005%, wt/wt) starting one week before exposure to 200 ppm PhIP for 61 days. PhIP treatment induced a approximately 8- to 16-fold increase in the mutation frequency (MF) in the colon. The induced MF was significantly lower in the cecum than in the proximal and distal colon (approximately 52 x 10(-5) vs. 100 x 10(-5), p < 0.008). CLA and DTT significantly reduced the PhIP-induced MF in the distal colon (p < 0.05) by 14% and 24%, respectively. The frequency of -1 frameshift mutations was lower in the distal colon of CLA- or DTT-treated rats. This protective effect was not observed in the cecum or in the proximal colon. In contrast, the PhIP-induced MF in the cecum (specifically, the frequency of -1 frameshifts and GC-->TA transversions) was elevated by 43% after treatment with CLA. In conclusion, CLA and DTT modulate PhIP-induced mutagenesis in a tissue-specific manner, and different modulation pathways are employed by CLA and DTT.

Identification of single nucleotide polymorphisms in human DNA repair genes.

Variation in gene coding sequence represents a significant factor in predisposition to disease, including cancer. Variants of some DNA repair genes (e.g. MLH1, MSH2 and MSH6) are known to predispose to cancer. We identified single nucleotide polymorphisms (SNPs) in five DNA repair genes in 142 healthy individuals using a DNA sequencing protocol optimized for the direct detection of single nucleotide polymorphisms. This approach, called the heterozygote sequencing protocol (HSP), enables moderate-scale population surveys of SNPs. HSP uses fluorescently tagged primers and exploits the large dynamic range and low background of automated fluorescent sequencing. HSP may be used for any sequence that can be amplified by PCR. A total of 12 SNP variants in MGMT, ERCC1, CDK7, CCNH and XRCC4 were identified, 11 at polymorphic frequencies, with an average frequency of 0.22 (95% confidence interval 0.20-0.24). Among the 82 individuals for whom complete SNP profiles were available, no one person carried the GenBank reference sequence for all five genes. The extensive heterogeneity observed in these five genes is intriguing. All variants are in Hardy-Weinberg equilibrium, although the meaning of this equilibrium is unclear. Using this approach, possible associations of sequence variation, and hence of variation in DNA repair, with disease risk can be assessed.

Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice.

The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations. Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed. Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment. We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice. A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups. While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not. The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007). The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI. The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C

Identification of genes responsive to BPDE treatment in HeLa cells using cDNA expression assays.

Genotoxic stresses induce cellular responses that can be observed at the level of gene expression. We have studied changes in gene expression following BPDE exposure in HeLa cells by using a cDNA expression array of 597 human genes. After a 53-hr exposure to 0.4 microM BPDE, nine genes were upregulated. The protein products of these genes are: fos-related antigen 2, apoptotic cysteine protease MCH4, DB1 (zinc finger protein 91), transcription factor ETR103, integrin alpha, interleukin-4, interleukin-6, 23-kDa highly basic protein, and ribosomal protein S9. We observed the downregulation of gene expression of three genes: heat-shock protein 27, DNA-binding protein TAX, and NADH-ubiquinone oxidoreductase B18 subunit. These results suggest unknown functions or regulatory circuits for several of the responsive genes and demonstrate the complexity of cellular responses to genotoxic insults.

Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test.

A significant fraction of human cancers are thought to have a genetic component and several lines of evidence suggest that deficiencies in DNA repair may be a contributing factor. Little is known, however, about the frequency and distribution of variants of DNA repair genes in the general human population. The protein truncation test (PTT) was used to screen 136 healthy volunteers for protein-truncating variants of 10 DNA repair genes: APE, CDK7, ERCC1, WAF1, HOGG1, MGMT, POLB, UNG, HAAG, and CCNH. This sample consisted of 41males (30%) and 95 females (70%) with an average age of 25.3 years, ranging from 17 to 60 years of age. No truncating mutations were found in the 10 genes examined in any of the subjects. The 95% confidence interval for a proportion of 0 over the 272 alleles examined per locus is 0-0.01. The calculated frequency of truncating mutations in each of these genes, among the general population, is thus less than 1%. Among the 10 genes tested in 136 people, a single sample had no PCR product for HAAG, even though PCR products were obtained on all other genes. Total RNA dot hybridization confirmed the presence of HAAG mRNA transcripts in this sample. Despite identification of this single DNA repair variant, these results indicate a low frequency of truncating mutations in DNA repair genes in the general population.

Tris(2,3-dibromopropyl)phosphate causes a gradient of mutations in the cortex and outer and inner medullas of the kidney of lacI transgenic rats.

Tris(2,3-dibromopropyl)phosphate (TDBP) is a kidney carcinogen in rats in which exposure results in tumors specifically in the outer medulla. We have previously shown that TDBP induces mutation in the rat kidney. Here we demonstrate that TDBP induces mutation in the kidney of the F344 Big Blue lacI transgenic rat in a gradient with the highest induction (6.4-fold) in the cortex and lowest induction (2.2-fold) in the inner medulla, when given at 2000 ppm in the feed for 45 days. Similar results were obtained at 100 ppm, although the gradient effect was less pronounced. Because exposure to TDBP results in increased cell proliferation in the outer medulla, our results suggest that tissue-specific targeting of TDBP-induced kidney tumors reflects the combination of cell proliferation and mutation induction. This is also the first known case when transgenic animals have been used to study mutation at the suborgan level.

Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue rats.

The cII assay provides an alternative choice to the lacI transgene for mutational studies involving Big Blue(R) transgenic mice and rats, or permits the evaluation of mutational responses in both genes. Here, we compare the mutational response of the cII gene from colon of Big Blue(R) F344 rats treated with a dietary mutagen and animal carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to those previously determined in the lacI transgene from colon of the same group of animals. A cursory inspection of PhIP-induced mutational spectra (MS) in cII and lacI suggests that the two transgenes respond differently to PhIP-induced mutation. However, a more thorough analysis of the MS in the two transgenes, including consideration of the number of mutational target sequences in each gene and nearest neighbor analyses of mutated nucleotides, indicates that PhIP-induced mutational specificity is similar in both genes. The evaluation of PhIP-induced mutational responses in these two transgenes serves as a model for intergenic mutational analyses.

Through a glass, darkly: reflections of mutation from lacI transgenic mice.

The study of mutational frequency (Mf) and specificity in aging Big Blue lacI transgenic mice provides a unique opportunity to determine mutation rates (MR) in vivo in different tissues. We found that MR are not static, but rather, vary with the age or developmental stage of the tissue. Although Mf increase more rapidly early in life, MR are actually lower in younger animals than in older animals. For example, we estimate that the changes in Mf are 4.9x10(-8) and 1.1 x 10(-8) mutations/base pair/month in the livers of younger mice (<1. 5 months old) and older mice (> or =1.5 months old), respectively (a 4-fold decrease), and that the MR are 3.9 x 10(-9) and 1.3 x 10(-7) mutations/base pair/cell division, respectively ( approximately 30-fold increase). These data also permit an estimate of the MR of GC --> AT transitions occurring at 5'-CpG-3' (CpG) dinucleotide sequences. Subsequently, the contribution of these transitions to age-related demethylation of genomic DNA can be evaluated. Finally, to better understand the origin of observed Mf, we consider the contribution of various factors, including DNA damage and repair, by constructing a descriptive mutational model. We then apply this model to estimate the efficiency of repair of deaminated 5-methylcytosine nucleosides occurring at CpG dinucleotide sequences, as well as the influence of the Msh2(-/-) DNA repair defect on overall DNA repair efficiency in Big Blue mice. We conclude that even slight changes in DNA repair efficiency could lead to significant increases in mutation frequencies, potentially contributing significantly to human pathogenesis, including cancer.