In vivo mutation at the human HPRT locus.

The molecular nature of mutations that arise in vivo at the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus can be determined. A wide variety of such mutations can be detected, including large and small deletions, frameshift mutations and single-base substitutions, as well as alterations that cause aberrant mRNA splicing. Here, we review the available information on mutations at this locus.

Mutation of human cells by kerosene soot.

The polycyclic aromatic hydrocarbon fraction of a kerosene soot induced forward mutation in human diploid lymphoblasts when coincubated with Sprague-Dawley rat liver postmitochondrial supernatant. Two components of the kerosene soot extract, benzo[a]pyrene (BP) and cyclopenta[cd]pyrene (CP), were also tested. TP was not mutagenic at the concentration found in the soot extract, although it was active at higher concentrations. The amount of CP present could account for approximately 8% of the total mutation observed with the soot. The results were compared to data obtained previously in a similar mutation assay in Salmonella typhimurium. The protocol described permits the facile assay of mutation at the hgprt locus in human lymphoblasts; such mutation is induced by compounds of complex mixtures requiring mixed-function oxygenase activity for metabolism to genetically active derivatives.

In vitro mutational spectrum of aflatoxin B1 in the human hypoxanthine guanine phosphoribosyltransferase gene.

The in vitro mutational spectrum of aflatoxin B1 (AFB1) in exon 3 of the human hypoxanthine guanine phosphoribosyltransferase gene in B-lymphoblasts was examined by a combination of polymerase chain reaction and denaturing gradient gel electrophoresis. The cell line used in this study contained an expression vector that produced high levels of human cytochrome P450 CYP1A1. CYP1A1 metabolizes AFB1 to form an epoxide intermediate which can react with DNA. About 1200 independent mutants were induced at the hypoxanthine guanine phosphoribosyltransferase locus by AFB1 and were selected en masse by addition of 6-thioguanine to the bulk culture. Two independent cultures were treated with AFB1. Polymerase chain reaction was used to amplify exon 3 from the complex mutant population, and denaturing gradient gel electrophoresis was used to separate wild-type DNA sequences from mutant sequences. Mutational hotspots were visible as discrete bands on the denaturing gradient gel. Scanning densitometry was used to determine the fraction of the complex population that was represented in each non-wild-type band. The bands containing the mutations were excised from the denaturing gradient gel and sequenced. In this way, the nature and frequency of mutational hotspots in a population of > 1000 mutants were determined. AFB1 produced one strong mutational hotspot in exon 3. Between 10 and 17% of the AFB1-induced mutants contained a single GC-->TA base substitution at base pair 209. This hotspot occurred in a GGGGGG sequence (the mutated base is underlined). This mutation was observed reproducibly in two independently treated cultures. Several other mutations were observed in only one culture but at a lower frequency. Our results are the first report of the mutational spectrum of AFB1 in a native human gene.

In vitro mutational specificity of cisplatin in the human hypoxanthine guanine phosphoribosyltransferase gene.

The in vitro mutational spectra of cisplatin [cis-diamminedichloroplatinum(II)] in exon 3 of the human hypoxanthine guanine phosphoribosyltransferase gene in B-lymphoblasts was examined by a combination of polymerase chain reaction and denaturing gradient gel electrophoresis. Several thousand independent mutants were induced at the hypoxanthine guanine phosphoribosyltransferase locus by cisplatin and were selected en masse by addition of 6-thioguanine to the bulk culture. Polymerase chain reaction was used to amplify exon 3 from the complex mutant population, and denaturing gradient gel electrophoresis was used to separate wild-type DNA sequences from mutant sequences. Mutational hotspots were visible as discrete bands on the denaturing gradient gel. Scanning densitometry was used to determine the fraction of the complex population represented by the novel bands. The mutant bands were excised from the denaturing gradient gel and sequenced. In this way, the nature and frequency of mutational hotspots in a population of several thousand mutants were determined. Cisplatin produced several mutational hotspots in exon 3. About 9-10% of the cisplatin-induced mutants had mutations in a GGGGGG sequence (base pairs 207-212). GC----AT substitutions at the second and third guanines in the 5'-GGGGGG-3' run made up about 2 and 4% of the induced mutants, respectively. About 4% of the induced mutants contained a GC----TA substitution at the sixth guanine. About 1% of the cisplatin-induced mutants had an AT----TA transversion in a TAGA sequence (base pair 271; mutated base is underlined). Our results are consistent with mutations occurring at GpG and ApG sites. These nucleotide sequences have been identified as the primary sites of cisplatin adduction.

Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells.

The formation and persistence of O6-ethylguanine, O4-ethylthymine, and O2-ethylthymine were quantitated in the genomic DNA of human lymphoblasts exposed to 1.0 mM N-ethyl-N-nitrosourea using immunoslot-blot. The three cell lines used included one which lacks O6-alkylguanine-DNA alkyltransferase, one deficient in nucleotide excision repair, and a third which is competent in both of these repair pathways. The activity of O6-alkylguanine-DNA alkyltransferase was further modulated with O6-benzylguanine, a specific inhibitor of this protein. Repair of the O-ethylated thymines was slow and not related to either DNA repair phenotype. O6-Ethylguanine was repaired with a half-life of about 8 h in cells which expressed both O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair functions. Cells expressing O6-alkylguanine-DNA alkyltransferase activity but lacking nucleotide excision repair showed only slow repair of O6-ethylguanine (half-life of O6-ethylguanine, 43 h), while cells lacking the alkyltransferase showed little or no repair of O6-ethylguanine regardless of nucleotide excision repair activity (half-lives of O6-ethylguanine, 53 to greater than 100 h). We conclude that O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair cooperate in the repair of O6-ethylguanine in human cells.

Specificity of mutations induced by the food-associated heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine in colon cancer cell lines defective in mismatch repair.

Recently, we have shown a hypermutable response to the food-associated heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP) in human cells defective in mismatch repair (MMR). These findings suggest that exogenous compounds such as PhIP may play an important role in the generation of tumors in MMR-defective individuals. The specificity of mutations induced by PhIP exposure at the endogenous HPRT locus was determined in cell lines defective in MMR to better understand the mutagenic effects of PhIP in MMR-defective individuals and to gain insight into the molecular mechanism of carcinogenesis induced by PhIP. Eighty-six induced HPRT mutants from two different cell lines were isolated and sequenced after exposure to 10 microM PhIP. Nineteen (22%) of these mutants contained G:C to T:A transversion mutations, consistent with the promutagenic adduct of PhIP at the C8 position of guanine miscoding with adenine. This level of PhIP-induced G:C to T:A transversions was approximately 4.5-fold higher than spontaneous G:C to T:A frequencies. Additionally, a hotspot for mutation was observed in a run of six guanines in HPRT exon 3, where a total of 23 (27%) of all PhIP-induced mutations occurred. These mutations consisted of transversions, transitions, and frameshift mutations. The increase in mutant frequency at this run of guanines corresponded to a 24-fold elevation above the spontaneous frequency in one cell line and a 3.3-fold increase in the other. These data suggest that PhIP may increase the risk of human carcinogenesis mediated by MMR by increasing mutations at runs of guanine residues. PhIP may thereby promote tumorigenesis by mutating growth-regulating genes that contain runs of guanines in their coding sequences, such as BAX, the insulin-like growth factor II receptor IGFIIR, and even the mismatch repair gene hMSH6.

Modulation of ethylnitrosourea-induced toxicity and mutagenicity in human cells by O6-benzylguanine.

We have examined the contributions of O6-alkylguanine-DNA alkyl-transferase (AGT) and nucleotide excision repair to the protection of human cells from the toxic and mutagenic effects of ethylnitrosourea. Three human lymphoblastoid cell lines were used: one which possesses both of these DNA repair pathways; one derived from a xeroderma pigmentosum complementation group A patient, which expresses AGT but is deficient in nucleotide excision repair; and a third which does not express AGT but is capable of excision repair. The level of active AGT in the cells was further modulated with the use of the AGT inhibitor, O6-benzylguanine. These cells were exposed to ethylnitrosourea in both the presence and absence of O6-benzylguanine, and population survival, growth, and mutagenesis at the hypoxanthine-guanine phosphoribosyl-transferase locus were measured. The results for all three measurements indicated that the lack of either AGT or nucleotide excision repair significantly impairs the ability of human cells to withstand DNA ethylation damage. Furthermore, the inhibition of AGT in xeroderma pigmentosum group A cells did not increase toxicity or mutagenicity, suggesting that AGT and nucleotide excision repair cooperate in the removal of DNA ethyl adducts. Related studies in our laboratory have shown that AGT and nucleotide excision repair are both necessary for the efficient removal of O6-ethyldeoxyguanosine.

Toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea in human cell lines with different DNA repair phenotypes.

We examined the toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea (ENU) in three Epstein-Barr virus-transformed human lymphoblastoid cell lines, each with a different DNA repair phenotype. One cell line lacks O6-alkylguanine-DNA alkyltransferase (AGT) activity; another, derived from a patient with xeroderma pigmentosum, complementation group A, lacks nucleotide exicision repair (NER) capability, and the third is competent in both repair functions. ENU-induced toxicity and mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase locus were increased to a similar degree relative to the repair-competent cells in both AGT-deficient and NER-deficient cells. We determined the mutational spectra for ENU by identifying DNA sequence changes at the hypoxanthine-guanine phosphoribosyltransferase locus in at least 26 clones resistant to 6-thioguanine from each cell line. Of the characterized mutations, 89% were single-base pair substitutions. Transitions and transversions were found at AT and GC base pairs in all three cell lines. The biggest difference within the spectra was in the rate of transitions at GC base pairs. Compared to the repair-competent cell line, this mutation was elevated about 8-fold in the AGT-deficient cells and about 3-fold in the NER-deficient cells. We conclude that both AGT and NER play an important role in protecting human cells from the toxic and mutagenic effects of ENU. Furthermore, the mutational spectra suggest that both of these repair systems participate in the repair of O6-ethylguanine adducts.

Molecular dosimetry of ethylene oxide: formation and persistence of 7-(2-hydroxyethyl)guanine in DNA following repeated exposures of rats and mice.

The formation of 7-(2-hydroxyethyl)guanine (7-HEG) in DNA of target and nontarget tissues was investigated in male B6C3F1 mice (20/group) and F344 rats (10/group) exposed to 0, 3, 10, 33, 100, or 300 (rats only) ppm ethylene oxide (ETO) by inhalation for 6 h/day for 4 weeks (5 days/week) and mice exposed to 100 ppm ETO for 1 or 3 days or 1, 2, or 4 weeks (5 days/week). The persistence of 7-HEG was studied in mice killed up to 7 days after cessation of the 4-week time-course study. In addition, the formation of O6-(2-hydroxyethyl)guanine and 3-(2-hydroxyethyl)adenine was evaluated in rats exposed to 300 ppm ETO. DNA samples from control and treated animals were analyzed for 7-HEG using neutral thermal hydrolysis, microconcentration, and high-performance liquid chromatography separation with fluorescence detection. Fluorescence-linked high-performance liquid chromatography was used for O6-(2-hydroxyethyl)guanine quantitation, and immunochromatography and gas chromatography-mass spectrometry were used for 3-(2-hydroxyethyl)adenine detection. Analysis of DNA from tissues of control mice and rats revealed the presence of peaks equivalent to 2-6 pmol 7-HEG/mg DNA. In mice exposed to 100 ppm ETO, 7-HEG accumulated to a similar extent in target and nontarget tissues, with adduct concentrations ranging from 17.5 +/- 3.0 (SE) (testis) to 32.9 +/- 1.9 (lung) pmol adduct/mg DNA after 4 weeks of exposure. Concurrent exposures of mice and rats to 100 ppm ETO for 4 weeks led to 2- to 3-fold lower concentrations of 7-HEG in mouse DNA in all tissues compared to rat DNA. 7-HEG disappeared slowly in a nearly linear fashion from the DNA of mouse kidney (t1/2 = 6.9 days) and rat brain and lung (t1/2 = 5.4-5.8 days), which was consistent with the loss of adduct mainly by chemical depurination. In contrast, a more rapid removal of 7-HEG from other mouse (t1/2 = 1.0-2.3 days) and rat (t1/2 = 2.9-4.8 days) tissues was consistent with adduct loss by depurination and DNA repair. Dose-response relationships for 7-HEG were nonlinear in both mice and rats, with the alkylating efficiency of ETO increasing at high exposures.(ABSTRACT TRUNCATED AT 400 WORDS)

Frequency and spectrum of ethylnitrosourea-induced mutation at the hprt and lacI loci in splenic lymphocytes of exposed lacI transgenic mice.

The development of mouse models with the endogenous hypoxanthine-guanine phosphoribosyl transferase (hprt) gene and lacI transgene as mutational targets provides an excellent opportunity to compare the mutant frequency (Mf) and types of mutations induced in vivo in different sequence contexts. To this end, a study was conducted to determine the Mfs and spectrum of mutations induced at these loci in splenic T cells from male B6C3F1 Big Blue mice (6 weeks old) exposed to N-ethyl-N-nitrosourea (ENU). Six weeks after i.p. injection of 40 mg ENU/kg, T cells were isolated from control (n = 7) and treated (n = 8) mice for the culture of hprt mutants and for the extraction of DNA and recovery of lacI mutants. Mutations in hprt exon 3 and in lacI were quantified and analyzed using published procedures (S. W. Kohler et al., Proc. Natl. Acad. Sci. USA, 88: 7958-7962, 1991; T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866-7870, 1992). In treated mice, the Mfs (average +/- SE) in hprt (6.0 +/- 0.2 x 10(-5)) and lacI (11.4 +/- 1.8 x 10(-5)) were approximately 16.2-fold (P = 0.006) and 3.4-fold (P = 0.009), respectively, above controls. However, the average induced Mfs (i.e., induced Mf = treatment Mf - background Mf) in hprt and lacI were similar, with the respective increases in Mf being 5.6 +/- 0.2 x 10(-5) and 8.0 +/- 2.3 x 10(-5) over background. Eleven of the 107 hprt mutants from treated Big Blue mice had mutations in exon 3, with 73% being substitutions at AxT bp. These data are similar to those observed in ENU-exposed nontransgenic B6C3F1 mice, in which 62 of 69 exon 3 mutations were substitutions at AxT bp (T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866-7870, 1992). For comparison, the sequences of the lacI genes in two to five mutants from each mouse were determined, and a total of 75 mutations (70 different mutations) was detected. In exposed mice, 55% (24 of 44) of the mutations in lacI were substitutions at AxT bp. In controls, substitutions at AT bp comprised only 20% of the recovered mutations in either hprt exon 3 (1 of 5) or lacI (5 of 26). These data indicate that the lacI mutation assay is less sensitive than the hprt assay for detecting increases in Mf induced by ENU exposure of mice as indicated by the lower relative increase in Mf in the lacI gene, but, given a 6-week expression time, the types of mutations induced by ENU in the transgene reflect those observed in the native transcribed gene.

Statistical test for the comparison of samples from mutational spectra.

The Monte Carlo estimate of the p value of the hypergeometric test is described and advocated for the testing of the hypothesis that different treatments induce the same mutational spectrum. The hypergeometric test is a generalization of Fisher's "exact" test for tables with more than two rows and two columns. Use of the test is demonstrated by the analysis of data from the characterization of nonsense mutations in the lacI gene of Escherichia coli. Unlike the chi-square test, the hypergeometric test remains valid when applied to sparse cross-classification tables. The hypergeometric test has the most discrimination power of any statistical test that could be employed routinely to compare samples from mutational spectra. Direct application of the hypergeometric test to large cross-classification tables is excessively computation intensive, but estimation of its p value via Monte Carlo techniques is practical.

Analysis of mutations occurring at the human hprt locus.

We have recently established a computerized database containing information on mutants at the human hypoxanthine guanine phosphoribosyl transferase (hprt) locus. The database contains sequence information on over 1000 mutants. We now present an analysis of the information in the database. 542 single base substitution mutants in the hprt coding region exist, and we have examined (1) the number of mutations and the number of mutable sites in each exon, (2) transcribed versus non-transcribed strand bias for mutations, (3) the frequency of the 5' and 3' nearest neighbors to a mutated base, and (4) the distribution of amino acid substitutions. The distribution of both DNA mutations and amino acid mutations was not uniform, several clusterings of mutations were observed and we propose several possible mechanisms to account for the hotspots. We also examined mRNA splicing mutants, mutants with small deletions, and frameshift mutants.

Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light.

In targeted mutagenesis of lambda phage by ultraviolet light, the mutations are caused by radiation-induced lesions in the phage DNA. Of 62 mutations in the lambda cI gene that were sequenced, 41 (63%) of the targeted mutations were transitions, with similar numbers of C X G to T X A and T X A to C X G base changes. The remaining 21 mutations were about equally divided among eight transversions, seven frameshifts (5 additions and 2 deletions), and six double events with either two nearby base changes or a base change and a nearby frameshift. Of the 62 mutations, 60 could be associated with -Pyr-Pyr- sequences in the DNA, sites of likely photoproducts. For more information on this point, lambda phage were irradiated with 313 nm light in the presence of acetophenone, for which the major photoproduct is reported to be the thymine-thymine cyclobutyl dimer, with no measurable Pyr(6-4)Pyo photoproducts. Of 22 mutations sequenced, 19 were transversions and only one was a transition, permitting the conclusion that thymine-thymine cyclobutyl dimers are not the primary cause of ultraviolet light-induced transitions. A consideration of all the data strongly suggests that Pyr(6-4)Pyo photoproducts are mutagenic lesions.

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase.

Polymerase chain reaction (PCR) was performed with two polymerases. Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenase). Both polymerases were used to amplify the same portion of the human 18S rRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenase did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (-)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures.

Sequence specificity of mutagenesis in the cI gene of bacteriophage lambda.

Studies of DNA base sequence alterations have shown that for every agent the mutagenic process is specific with respect to the types of base changes induced and the location of the changes in the DNA. Analysis of the types of mutations produced by mutagenic agents can provide insight into the mechanism of mutation and can suggest which DNA lesions may be involved in the actual mutagenic event. We have developed a system for the analysis of chemically induced base sequence alterations in the cI repressor gene of bacteriophage lambda using DNA sequencing techniques. To illustrate the utility of this type of analysis, we present the results obtained with ultraviolet light (UV). Irradiation of target DNA with UV alone, or UV followed by photoreactivating light (which removes dimers), produces mostly transitions at pyrimidine-pyrimidine sites. Conversely, irradiation with 313 nm light plus acetophenone (which produces only thymine dimers) produces mostly transversions at low efficiency. This and other evidence suggests that the actual premutagenic UV lesion in E. coli may not be pyrimidine-pyrimidine dimers, but rather pyr(6-4)pyo photoproducts.

In vivo mutations in human blood cells: biomarkers for molecular epidemiology.

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) < hprt (5 x 10(-6)) = GPA (10 x 10(-6)) < HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. II. hprt mRNA expression and cDNA sequence analysis of induced mutants.

Mutants at the hprt locus isolated after treatment of cells of the human lymphoblastoid cell line TK6 with X rays were examined by Northern blot and cDNA sequence analysis. In previous work, Southern blot analysis showed that approximately 25% of the mutants isolated from cultures treated with X rays displayed restriction fragment patterns indistinguishable from wild type. In addition, 38 and 48% of the mutants isolated from cultures treated with X rays under two radioprotective conditions, hypoxia or 25 mM cysteamine, respectively, had normal restriction fragment patterns. In the work presented here, Northern blot and DNA sequence analyses were used to characterize these mutants further. Mutants were classified as having normal size and amount of hprt mRNA, reduced amount or undetectable mRNA, or abnormal size message. Mutants that expressed hprt mRNA were sequenced after reverse transcription and PCR amplification. Sequence analysis is reported for 7 mutants from cultures treated in the absence of protection, 11 from cultures treated under hypoxic conditions, and 11 from cultures irradiated in cysteamine. Striking differences among the three treatment groups were not apparent. All types of mutations at both AT and GC base pairs were observed, including transitions, transversions, small deletions, and insertions. Several mutations affected RNA splicing, leading to exon skipping or inclusion of intron sequences in the final message. Approximately half (14/29) of the mutants sequenced had additions or deletions of one to several nucleotides. Also, 3/29 involved tandem DNA base changes (GG-->TT, C-->AA, AA-->CG). These observations are consistent with a mechanism involving the induction of noncoding or synthesis-blocking lesions that result in polymerase slippage or error-prone bypass synthesis. In addition, potential hotspot sites for mutation by X rays were discovered. At one site in exon 3, the same complex mutation, consisting of a G-->T transversion and a nearby six-base deletion, was detected in three independent mutants. Another mutant had a G-->C transversion at the same base, but without the deletion. At another site in exon 8, three mutations occurred at a run of three consecutive cytosines; these included a -C, a -CC, and a C-->AA. Also in exon 8, two mutations (+T, T-->C) occurred at two consecutive thymines.

Cytogenetic analysis of spontaneous and 2-cyanoethylene oxide-induced tk-/- mutants in TK6 human lymphoblastoid cultures.

Two phenotypic classes of selectable tk-/- mutants have been isolated from the TK6 human lymphoblast cell line; one has a normal growth rate (tkn) relative to the parental cell line; the other has a slow growth rate (tks). Complete karyotypes of metaphase chromosomes were prepared and analyzed from 16 tks mutants (eight spontaneous and eight 2-cyanoethylene oxide [CNEtO]-induced), two spontaneous tkn mutants, and the parental TK6 cell line. Southern blot analysis of these tk-/- mutants indicated that all had lost a 14.8 kb polymorphic band corresponding to the active tk allele. No chromosome abnormalities with respect to the parental cell line TK6 were observed in eight spontaneous tks mutants. Chromosome abnormalities that may have been related to CNEtO treatment were observed in four of eight CNEtO mutants. However, chromosome 17, containing the tk locus in man, was cytologically normal with respect to the parental TK6 cell line in 15 of 16 tks mutants. A visible abnormality of chromosome 17 was present in one CNEtO-induced tks mutant. The abnormality was a duplication of the long arm of the chromosome 17, with break points at q11 and q21. The latter break point is close to the site of the tk locus, suggesting that the aberration observed may be associated with tk-/- phenotype. These observations contrast with the relatively high incidence (greater than or equal to 59%) of chromosome 11 abnormalities reported in tks (small colony) mouse lymphoma L5178Y/TK +/- mutants.

Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells.

Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10(-7) and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced-1 frameshift reversion in the GGGGGGG sequence was approximately 500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells.

Relative sensitivity of the endogenous hprt gene and lacI transgene in ENU-treated Big Blue B6C3F1 mice.

Three-week-old Big Blue (BB) B6C3F1 mice were given a single i.p. injection of ENU. Three weeks later, splenic T cells were isolated from each animal by ficoll gradient centrifugation and divided into two samples. One sample was cultured to measure hprt- mutation and the other was used to extract DNA for lacI- analysis. T cells from BB mice exposed to 0, 4.5, 13.5, and 40 mg ENU/kg (9 or 10 animals per group) displayed dose-related increases in the frequency of both hprt- and lacI- mutations. Within each treatment group, the ENU-induced mutation frequency (average observed mutation frequency minus average control frequency) was remarkably similar at the two loci. This suggests that treatments that increase mutation frequency at the endogenous hprt gene also produce similar incremental increases at the BB lacI transgene. However, because of the ten-fold higher spontaneous mutation rate at lacI, the fold-increase over background produced by ENU at this locus was significantly less than the fold-increase produced at hprt. For example, the 4.5 mg ENU/kg treatment produced a 5.2-fold increase above background at hprt (P = 0.001), whereas only a 1.5-fold increase was produced at lacI (P = 0.140). Consequently, mutagenic insults that produce up to a fivefold increase in mutation frequency at an endogenous locus may be difficult to detect at the lacI transgene. Finally, the ENU-induced response at hprt in BB mice was identical to that in generic B6C3F1 mice, suggesting that there are no inherent differences between transgenic and normal mice in their response to this mutagenic agent.