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.

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.

An analysis of acute changes in interleukin-6 levels after treatment of hepatitis C with consensus interferon.

Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

Human HPRT mutant database: software for data entry and retrieval.

We have developed a computer database containing information on over 1,000 human hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants. Both published and unpublished data are present. The database itself is maintained in a dBASE format (.DBF) and we provide a set of programs to examine and extract information from the database. A program to input information into the database is also supplied. The database and programs are available directly from us or via remote FTP (file transfer protocol) using BITNET/INTERNET. All programs require an IBM-compatible computer, the MS-DOS operating system (version 3.3 or greater), and a hard disk with about 5 megabytes of free disk space. The purpose of the database is 1) to allow investigators to contribute their HPRT mutants directly to the database in a standardized fashion, and 2) to allow access to the entire database with a set of programs that allows manipulation and extraction of data. For example, using our programs it is possible to i) order the database by base pair position, ii) examine only information regarding mutagenesis by a particular agent, iii) search for a particular author, iv) create a report which contains selected portions of the database, the report can be printed or saved as a file. The database will be updated every several months and distributed.

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli.

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT- human lymphoblast colonies induced by eight repetitive 150 microM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli, DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism.

Formaldehyde mutagenesis and formation of DNA-protein crosslinks in human lymphoblasts in vitro.

Human lymphoblasts were exposed in vitro to various concentrations of formaldehyde (HCHO) in single and multiple treatment regimens to determine relative mutagenic efficiency. Single treatments of HCHO (0-150 microM X 2 h) resulted in a nonlinear increase in induced mutant fraction at the thymidine kinase locus with increasing slope at concentrations above 125 microM. Only HCHO exposures of 125 microM X 2 h or greater produced significant effects on the growth rate of the lymphoblasts. Cultures were also exposed to either three treatments of 50 microM X 2 h, five treatments of 30 microM X 2 h, or ten treatments of 15 microM X 2 h; multiple treatments were administered on different days. These multiple treatments resulted in increases in mutant fraction, although their combined effect was less than a single treatment of equivalent concentration X time (150 microM X 2 h). Exposure of lymphoblasts to four treatments of 150 microM X 2 h HCHO failed to induce mutations at the ouabain resistance locus. Cultures of lymphoblasts receiving a single treatment of HCHO (0-600 microM X 2 h) were analyzed by the alkaline elution technique to detect the presence of DNA-protein crosslinks. HCHO treatment resulted in a significant nonlinear increase in DNA-protein crosslinks at concentrations greater than 50 microM X 2 h, which correlated with the onset of significant toxicity in this cell line. Holding the culture for 24 h resulted in complete removal of the crosslinks. These data indicate that both the induction of mutations and the formation of DNA-protein crosslinks by HCHO are nonlinear functions in human lymphoblasts and occur at overlapping concentration ranges.

Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.

We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine (guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hprt- mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-gradient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprt- mutant frequency of 7.3 x 10(-5) in splenic T cells, approximately 35-fold above background levels. Sixty-nine of the 521 Hprt- mutants analyzed contained mutations in exon 3 (13%). Transversions and transitions at A.T base pairs dominated the spectrum; 62 of the 69 exon 3 mutations were at A.T base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at A.T bases) were located on the nontranscribed strand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G.C base pair. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse Hprt- T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.