Mutagenesis vol. 19 no. 3 pp. 215-222,
May 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
In vivo mutagenicity and mutation spectrum in the bone marrow and testes of B6C3F1 lacI transgenic mice following inhalation exposure to ethylene oxide
CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709, USA 1Present address: Genetic and Cellular Toxicology, Merck Research Laboratories WP45-324, West Point, PA 19486, USA 2Present address: E.I. du Pont de Nemours and Co., Duport Haskell Laboratory for Health and Environmental Sciences, PO Box 50, 1090 Elkton Road, Newark, DE 19711, USA 3Present address: AstraZeneca R&D Sodertalje, S-151 85 Sodertalje, Sweden 4Present address: Director, Environmental Carcinogenesis Division, US Environmental Protection Agency NHEERL, MD-68 Research Triangle Park, NC 27711, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
The lacI mutant frequency and mutation spectrum were determined in the bone marrow and testes of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of male transgenic lacI B6C3F1 mice were exposed to 0, 25, 50, 100 or 200 p.p.m. EO for up to 48 weeks (6 h/day, 5 days/week) and were killed at 12, 24 or 48 weeks of EO exposure for determination of lacI mutant frequency. In the bone marrow, the lacI mutant frequency was significantly increased at the two highest exposure levels (100 and 200 p.p.m.) and at the 48 week exposure time point. The shape of the exposure–response curve for lacI mutant frequency in the bone marrow was non-linear. DNA sequence analysis of the bone marrow mutation spectrum revealed that only AT
TA transversions occurred at an increased frequency in EO-exposed mice: 25.4% in EO-exposed mice for 48 weeks (200 p.p.m.) compared with 1.4% in air controls. In testes, the lacI mutant frequency was increased at a single exposure level of 200 p.p.m. for 24 weeks. At 48 weeks, the lacI mutant frequency in testes was significantly increased to an equal degree at 25, 50 and 100 p.p.m. EO but not at 200 p.p.m. Analysis of the testes mutation spectrum in air control mice and in mice exposed to 200 p.p.m. EO for 48 weeks revealed that no single mutational type occurred at an increased frequency. In the testes, there was a small increase across all mutational types that was sufficient to increase the overall lacI mutation frequency although not significant individually. The mutation spectrum in testes of EO-exposed mice also revealed that the increased lacI mutant frequency observed at 25 or 50 p.p.m. EO was not due to an increase in mutant siblings (clonality). These data demonstrate that inhalation exposure to EO for up to 48 weeks produces distinct mutagenic responses in bone marrow and testes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
Ethylene oxide (EO) (CAS no. 75-21-8) is a major industrial intermediate and sterilant gas regulated by the US Environmental Protection Agency as a hazardous air pollutant under the Clean Air Act (US Environmental Protection Agency, 1991
). EO is a direct-acting mutagen that reacts with nucleophilic molecules, including DNA, causing gene mutation and chromosome deletions in somatic cells (Dellarco et al., 1990; IARC, 1994). Intraperitoneal injection of EO produces increases in bone marrow micronuclei in rats and mice and is mutagenic at Hprt in the T lymphocytes of B6C3F1 mice (Appelgren et al., 1978; Walker and Skopek, 1993). Inhaled EO induces an increased frequency of lacI mutants recovered from lung and Hprt mutant T lymphocytes in mice and rats (Sisk et al., 1997; Walker et al., 1997; Tates et al., 1999; van Sittert et al., 2000; Walker et al., 2000). Increased levels of sister chromatid exchange also occur in peripheral lymphocytes of rodents and monkeys after inhalation of EO (Yager and Benz, 1982; Kligerman et al., 1983; Lynch et al., 1984; Lewis et al., 1986; Ribiero et al., 1987). In biomonitoring studies, cohorts of EO-exposed workers show increased frequencies of several chromosomal aberration end-points relative to unexposed workers (IARC, 1994). A review on the genotoxic effects of EO in humans has recently been published (Kolman et al., 2002).
In 2 year inhalation bioassays, EO induced exposure-related increases in gliomas, peritoneal mesotheliomas and mononuclear leukemias in F-344 rats and lymphomas and adenomas and carcinomas of the lung, harderian gland and mammary gland in B6C3F1 mice (Snellings et al., 1984; National Toxicology Program, 1988). In human epidemiological studies reviewed by the International Agency for Research on Cancer, mortality from lymphatic and hematopoietic cancer was marginally elevated (IARC, 1994). IARC (1994) has classified EO as a Group 1 chemical (carcinogenic to humans). This classification was based on several lines of evidence, including limited data in humans for carcinogenicity, sufficient evidence in experimental animals and surrogate biomarkers of cancer. These surrogate biomarkers of cancer included formation of DNA adducts and genotoxicity in EO-exposed humans and rodents. The National Toxicology Program classified EO as ‘known to be a human carcinogen’ based on sufficient evidence of carcinogenicity from studies in humans, which included combination epidemiological and mechanistic investigations indicating a causal relationship between exposure to EO and human cancer (National Toxicology Program, 2001).
Transgenic animal mutation models permit the determination and molecular analysis of in vivo mutation in marker genes from the tissues of exposed animals (Mirsalis et al., 1994). The induction of mutations can be determined in numerous tissues with respect to exposure, dose to target tissue, DNA adduct levels and additional genotoxicity end-points. Inhalation exposure of B6C3F1 lacI transgenic mice to EO for 4 weeks at 50, 100 or 200 p.p.m. did not induce a significant increase in lacI mutant frequency in bone marrow, spleen cells or DNA isolated from preparations of testicular seminiferous tubules (Sisk et al., 1997). However, in the lungs, a known target tissue for EO-induced tumors in mice (National Toxicology Program, 1988), a significant increase in lacI mutant frequency was observed (Sisk et al., 1997). In the same study, EO induced a significant dose-dependent increase in the frequency of Hprt mutant T lymphocytes isolated from the spleen of mice (Walker et al., 1997). We hypothesized that the increase in Hprt mutant T cells, but not in lacI mutation, was due to the poor recovery of large deletions (>1.0 kb) with 
phage based shuttle vector systems (Gossen et al., 1995; Sisk et al., 1997). We used these differential responses at Hprt and lacI to suggest that the primary mode of in vivo genotoxicity induced by short-term exposures to EO was likely due to large deletions and chromosomal mutations not recovered as mutations in phage based shuttle vector systems.
In addition to somatic cell genotoxicity, EO can induce transmissible genetic damage in germ cells. Intraperitoneal injection of EO induced dominant lethal mutations and reciprocal translocations in post-meiotic germ cells from male mice (Generoso et al., 1980). On inhalation exposure to EO, dominant lethals and heritable translocations increased in a non-linear, exposure-related manner at high exposure levels (>167 p.p.m.) (Generoso et al., 1986, 1990). An initial US Environmental Protection Agency genetic risk assessment of EO using these data was presented in a series of papers (Dellarco et al., 1990; Generoso et al., 1990; Rhomberg et al., 1990). This initial EO genetic risk assessment was re-examined, taking into account aspects of effective dose to the target tissue, mechanisms of chromosomal alterations and the shape of the dose–response curve at low doses (Natarajan et al., 1995; Preston et al., 1996). These biologically based genetic risk assessments identified data gaps and recommended that components of a genetic risk assessment for genotoxic chemicals such as EO should include dose-dependent biological response data using exposures that encompass complete cycles of spermatogenesis and measurements of point mutations, deletions and chromosomal alterations in target cell populations (Preston et al., 1996). Biologically based cancer risk assessments for EO have used hemoglobin or DNA adducts as measures of internal dose and Hprt mutant T lymphocytes as measures of response to predict a cancer unit risk for EO (Tates et al., 1999; Their and Bolt, 2000; van Sittert et al., 2000).
In this article we report the in vivo lacI mutant frequencies determined in bone marrow and testes of B6C3F1 lacI transgenic mice exposed to EO by inhalation. We used the lacI system in the present study to assess the induction of point mutations under long-term EO exposure conditions that approximate the chronic exposure conditions used in the EO cancer bioassay and would permit the assessment of mutagenicity over complete cycles of spermatogenesis (Snellings et al., 1984; National Toxicology Program, 1988). Groups of B6C3F1 mice were exposed in whole body chambers to EO at 0, 25, 50, 100 or 200 p.p.m. for 6 h/day, 5 days/week for 12, 24 or 48 weeks. The lacI mutant frequency was quantified at every exposure level to determine the shape of the exposure–response curve for EO-induced lacI mutant frequency. The mutation spectrum by DNA sequence analysis was determined to identify mechanisms of mutational change in the lacI gene recovered from bone marrow and testes. The mutation spectrum was also used to assess the contribution of independent mutational events to the lacI mutant frequency as part of the data needed to assess the shape of the exposure–response curve for EO-induced lacI mutations.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
Chemicals
Chemicals used for the present studies were purchased as follows: ethylene oxide (purity 99%), ARC Chemical Division, Balchem Corp., Slate Hill, NY; proteinase K, Sigma; phenol:chloroform, Amresco, St Louis, MO; 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal), Gold Biotechnologies, St Louis, MO; dimethylformamide, Sigma-Aldrich, St Louis, MO.
Animals
Male B6C3F1 lacI transgenic mice (lacI mice) (Big BlueTM) 4–6 weeks of age were purchased from Stratagene Cloning Systems (Taconic Farms, Germantown, NY) and acclimated for 10–14 days prior to exposure. Animals were randomized by weight and then for each time point examined (12, 24 and 48 weeks), groups of five or six mice per EO exposure group or air controls were placed in 1 m3 exposure chambers. All animal use was conducted in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, approved by the Animal Care and Use Committee of the CIIT Centers for Health Research (CIIT) and consistent with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
EO exposure levels
EO exposure levels of 25, 50, 100 or 200 p.p.m. were used in the present study. These exposure levels were chosen based on the NTP carcinogenicity bioassay using B6C3F1 mice exposed to 50 or 100 p.p.m. EO for 2 years (National Toxicology Program, 1988), the frequency of transmissible heritable translocations at levels of EO = 167 p.p.m. (Generoso et al., 1990), and EO-induced DNA adduct levels (N7-hydroxyethylguanine, HO-ethyl-N7-G) in mice exposed to =100 p.p.m. EO (Walker et al., 1992). In mice, inhalation exposure levels of 25–200 p.p.m. EO for 4 weeks are in the linear range for the formation of HO-ethyl-N7-G DNA adducts (Walker et al., 1992).
Inhalation exposures to ethylene oxide
Four groups of male lacI mice, 6- to 8-weeks-old at exposure start, were exposed to constant whole body target exposures of 25, 50, 100 or 200 p.p.m. EO, respectively, for up to 48 weeks (6 h/day, 5 days/week). The air control mice were exposed to clean air in a separate chamber in the same suite of rooms. The number of mice in each group (n) was as follows: air control, n = 17; 25 p.p.m., n = 16; 50 p.p.m., n = 16; 100 p.p.m., n = 17; 200 p.p.m., n = 16. The animals in each exposure group and in the air control group were housed individually in hanging steel cages inside a 1 m3 inhalation chamber (H1000; Lab Products, Maywood, NJ). The mice were given access to water but deprived of food during the 6 h exposure period. After each 6 h exposure, the mice were fed (NIH-07 certified feed) until the next exposure period.
EO concentrations in the exposure chambers were monitored with an infrared spectrophotometer (MIRAN 1A; The Foxboro Co., Foxboro, MA). During the initial phase of the study, pressure and relative humidity interfered with the air control chamber concentration monitoring (baseline drift). Therefore, a gas chromatograph (Model 5890 Series II; Hewlett-Packard, Palo Alto, CA) equipped with a flame ionization detector was used to concurrently monitor the air control chamber. Concentrations were monitored at least once per hour from each of the five chambers.
Necropsy and tissue collection
Five or six animals from each exposure group (0, 25, 50, 100 or 200 p.p.m. EO) were killed 3 days after the final exposure day for the 12, 24 and 48 week time points. The mice were asphyxiated using carbon dioxide and exsanguinated by cardiac puncture. Bone marrow cells were collected by flushing of the bone marrow with 2 ml of phosphate-buffered saline (PBS), pelleted by centrifugation and then flash frozen in liquid nitrogen. Testes were excised, a small sample for chromosomal analysis was collected and the remaining tissue was flash frozen in liquid nitrogen. All organs and bone marrow excised from the animals were subsequently stored at –80°C until DNA extraction.
LacI mutant frequency determinations in tissues of transgenic mice
Genomic DNA was prepared from frozen tissues according to the instructional manual for the Big BlueTM DNA isolation kit; solutions, buffers and extraction procedures are described in detail in the instruction manual (Stratagene, La Jolla, CA). For bone marrow, the frozen cell pellet was thawed by resuspension in DNA extraction cell lysis buffer, homogenized and subjected to proteinase digestion. The DNA was then extracted. Frozen testes were thawed in cell lysis buffer, the epithelial capsule was removed and the seminiferous tubules in aggregate were excised and incubated in a freshly prepared collagenase solution (Provost and Short, 1994). The seminiferous tubules were gently pelleted and subjected to proteinase digestion and DNA extraction. Testes DNA referred to here and throughout this manuscript is the DNA extracted from seminiferous tubules. After ethanol precipitation, the DNA was resuspended in 50–200 µl of buffer and allowed to stand at room temperature overnight before storage at 4°C.
The shuttle vector containing the lacI transgene was recovered from bulk genomic DNA with phage packaging extract (Transpack; Stratagene) used according to the manufacturer’s instructions. Packaged phage were preadsorbed to Escherichia coli SCS-8 cells for 20 min at 37°C, mixed with prewarmed NZY top agar [N-Z-Amine A (Casein Enzymatic Hydrolysate) and Yeast Extract] containing 1.3 mg/ml X-gal and poured into 25 cm2 square plates containing NZY agar. Plates were incubated overnight at 37°C.
The total number of colorless phage plaques was estimated for each plate by counting three 1 cm2 sections, averaging these numbers and multiplying by the area of the plate. Blue lacI plaques were counted and picked into individual tubes containing 500 µl of SM buffer and 50 µl of chloroform for further characterization. All mutant plaques were confirmed by restreaking on an SCS-8 bacterial lawn on X-gal-containing NZY agar. Mosaic plaques occurred rarely and were not included in the analysis since sectored plaques are likely to be the result of lacI ex vivo mutation during E.coli replication or repair.
The lacI mutant frequencies were calculated by dividing the number of confirmed mutant plaques by the total number of plaques analyzed. Following published recommendations on the conduct of the lacI assay in mice (Piegorsch et al. 1994; Callahan and Short, 1995), between five and six animals per group per time point were used to determine the lacI mutant frequency. Approximately 200 000 plaques from each animal were examined from either bone marrow or testes DNA.
Statistical analysis of mutant frequency in air control and EO-exposed mice
The statistical analyses were done using JMP® statistical software package (SAS Institute, Cary, NC). The lacI mutant frequency was log-transformed to normalize variance among samples. Student’s t-test (two-tailed) was used to determine the statistical significance of mutant frequency in EO-exposed mice relative to air controls in bone marrow and in testes from EO-treated mice compared with air controls (Piegorsch et al., 1994; Callahan and Short, 1995). A value of P < 0.05 was considered significant.
DNA sequence analysis of lacI mutants
The lacI gene was sequenced using an ABI cycle sequencing kit (ABI PrismTM terminator cycle sequencing ready reaction kit with Amplitaq® DNA polymerase, Fragment Stoffel; Applied Biosystems, Foster City, CA) in an ABI 373A DNA Sequencer (Applied Biosystems) using AutoAssembler 1.3 software (Applied Biosystems) for data analysis. The complete lacI gene was sequenced for each mutant isolated from each of five or six animals used to determine the lacI mutant frequency. In most samples, 5–10 mutants used in the lacI mutant frequency determination for each animal were also used to develop the mutation spectrum. From the bone marrow, lacI mutants for DNA sequence analysis were isolated from air control mice and mice exposed for 48 weeks to 200 p.p.m. EO. From the testes, lacI mutants for DNA sequence analysis were isolated from air control mice and mice exposed for 48 weeks to 25 and 50 p.p.m. EO. All DNA sequences shown represent the lacI coding strand in the 5'3' direction with the first G in the GTG start codon as base 29 (Farabaugh et al., 1978).
Statistical analysis of mutational spectrum in air control and EO-exposed mice
The frequency and percentage of each mutational type among the air control and EO-exposed groups was compiled for statistical analyses. The mutation frequency for the air control and EO exposure groups was based on DNA sequence analysis and calculated using the mutant frequency and the frequency of independent mutational events in each DNA sample, i.e. adjusting for mutant siblings (Recio and Meyer, 1995; Saranko et al., 2001). The mutation frequency for each mutational type was compared using recommended methods (Carr and Gorelick, 1996): log-transformation of the mutation frequency of each mutational type and Student’s t-test (two-tailed, P < 0.05). Fishers exact test (
< 0.05) was also used to statistically compare each of the mutational types that contributed to the mutation frequency for air control and EO-exposed mice (Recio et al., 1998). All statistical analyses were done using JMP® statistical software package (SAS Institute).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
EO exposure levels and conditions
The target exposure levels for each EO exposure chamber and for air controls were 0, 25, 50, 100 and 200 p.p.m., 6 h/day, 5 days/week for up to 48 weeks. Actual EO concentrations (in p.p.m. ± SD) in each exposure chamber for the entire 48 week exposure period were 0 ± 0, 25 ± 2, 50 ± 2, 100 ± 3 and 199 ± 4 for the target concentrations of 0, 25, 50, 100 and 200 p.p.m., respectively. Temperature and relative humidity for chambers monitored for the entire 48 week exposure period was between 71.9 and 72.3°C and 43 and 44%, respectively. The air control mice were exposed to clean air of the same temperature, relative humidity and air flow as delivered to the EO-exposed animals.
Determination of lacI mutant frequency in bone marrow of mice
The length of EO exposure (12, 24 or 48 weeks) and lacI mutant frequencies for the bone marrow at each of the exposure levels examined are shown in Table I. The lacI mutant frequency in bone marrow was significantly increased compared with air controls at the 48 week time point for mice exposed to 100 and 200 p.p.m. EO, with respective mutant frequency values of 7.3 ± 2.4 x 10–5 compared with 14.1 ± 7.3 x 10–5 and 30.3 ± 11.9 x 10–5. There was a non-linear increase in lacI mutant frequency in the bone marrow at 48 weeks with increasing exposure levels of EO (Fig. 1).
|
|
DNA sequence analysis of lacI mutants from the bone marrow of air control and EO-exposed mice
DNA sequence analysis was performed on lacI mutants isolated from air controls and EO-exposed mice to determine the mutational basis for the observed increase in mutant frequency and the contribution of independent mutations to the mutant frequency (Table II). The lacI mutant and lacI mutation frequency (adjusted for mutant siblings) for air controls at 48 weeks were 7.3 x 10–5 and 6.9 x 10–5, respectively, with 93% unique mutations (7% contribution of mutant siblings). For mice exposed to EO at 200 p.p.m. for 48 weeks, the lacI mutant and lacI mutation frequency were 30.3 x 10–5 and 25.3 x 10–5, respectively, with 83% unique mutations (17% contribution of mutant siblings) (Table II). The mutation frequency was used with the mutation spectrum to determine the contribution of individual mutational types.
|
Mutational spectrum in bone marrow of air control and EO-exposed mice
From the air control group at 48 weeks, 75 lacI mutants isolated from the bone marrow were sequenced and 70 unique mutations identified 7% mutant siblings. Mutational types and the frequency of each are summarized in Table II. Detailed mutation spectra for each lacI mutant analyzed from bone marrow are available at http://www.mutage.oupjournals.org/. Single base substitutions accounted for 91% (64/70) of the background mutations in air control mice, with the remaining 9% due to small deletions, insertions or tandem base changes. The greatest frequency of base pair substitutions occurred at GC base pairs, constituting 78% (54/70) of the background base substitutions, with 57% (40/70) due to GC
AT transitions. Among the GCAT transitions in air controls, 78% (31/40) occurred at 5'-CpG-3' dinucleotides. GCAT transitions at CpG nucleotides are preferentially involved as background mutations in this system in all tissues examined (Kohler et al., 1991; Provost et al., 1993; Sisk et al., 1994; de Boer et al., 1998) and likely result from deamination of 5-methylcytosine to thymine (Bird, 1986). Among the background mutations in air control mice, 14% (10/70) occurred at AT base pairs; 1.4% (1/70) of the mutations were ATTA transversions.
From the bone marrow of the group exposed to EO at 200 p.p.m., 66 mutants were sequenced and 55 unique mutations identified 17% mutant siblings. This group was selected for sequencing because it exhibited the highest overall mutant frequency in the bone marrow among the EO-exposed groups. Single base substitutions accounted for 90% (50/55) of the mutations in the EO-exposed group, with 49% (27/55) at GC base pairs and 41% (23/55) at AT base pairs. Among the 18 GCAT transitions, 44% (8/18) occurred at 5'-CpG-3' dinucleotides and 56% (10/18) did not. In the bone marrow of EO-exposed mice, 25.4% (14/55) of the lacI mutations isolated were due to ATTA transversions. In contrast, ATTA transversions occurred at a frequency of 1.4% (1/70) in air control mice.
The lacI mutation frequency (after adjusting for clonality) was significantly greater (P < 0.05) in EO-exposed mice (25.3 x 10–5) relative to air controls (6.9 x 10–5). To identify EO-specific mutations, the contribution of each mutational type to the mutation frequency was calculated and used to compare EO-exposed mice (200 p.p.m. for 48 weeks) to air controls. ATTA transversions occurred at a significantly increased frequency (P < 0.001) in the bone marrow of EO-exposed mice at 48 weeks of exposure. No other mutational types occurred at an increased frequency in EO-exposed mice compared with air controls. There were 10 sites of mutation in common between air control and EO-exposed mice.
Determination of lacI mutant frequency in testes of mice
The length of EO exposure (12, 24 or 48 weeks) and lacI mutant frequencies for the testes at each of the exposure levels examined are shown in Tables III and IV. The lacI mutant frequency in testes of EO-exposed mice was significantly increased at one exposure level of EO (200 p.p.m.) at 24 weeks and was increased in groups of mice exposed to 25, 50 or 100 p.p.m. for 48 weeks (P < 0.05) when compared with the lacI mutant frequency in the corresponding air controls. The increased lacI mutant frequency determined in testes from EO-exposed mice (25, 50 or 100 p.p.m.) was equivalent among these three exposure groups and 
2-fold greater than that determined for the air controls. The lacI mutant frequency in mice exposed to 200 p.p.m. for 48 weeks was not increased and was equivalent to that determined in air controls (Table IV). The lacI mutant frequency (± SD) determined in the testes of air controls (0 p.p.m.) and in mice exposed to 25, 50 or 100 p.p.m. for 48 weeks is plotted in Figure 2a.
|
|
|
Mutational spectrum in testes of air control and EO-exposed mice
From the air control group at 48 weeks, 60 lacI mutants isolated from the testes were sequenced from the 48 week air control group and 44 unique mutations were identified, 26% mutant siblings. Mutational types and the frequency of each are summarized in Table V. Detailed mutation spectra for each lacI mutant analyzed from testes are available at http://www. mutage.oupjournals.org/. Single base substitutions accounted for 84% (37/44) of the background mutations in air control mice, with the remaining 16% due to small deletions, insertions or tandem base changes. Similar to the bone marrow, the greatest frequency of base pair substitutions occurred at GC base pairs, constituting 77% (34/44) of the background base substitutions, with 39% (17/44) due to GC
AT transitions. Among the GCAT transitions, 82% (31/40) occurred at 5'-CpG-3' dinucleotides. Among the background mutations in testes, 7% (3/44) occurred at AT base pairs; all three were ATGC transitions.
|
As in air controls, single base substitutions accounted for 85% (77/89) of the mutations in the EO-exposed group, with 75% (67/89) at GC base pairs, 10% (9/89) at AT base pairs and 15% (13/89) involving multibase alterations (Table V). Among the 43 GC
AT transitions, 81% (35/43) occurred at 5'-CpG-3' dinucleotides and 19% (8/43) did not. Of the mutations isolated from the testes of EO-exposed mice, 10 (9/89) occurred at AT base pairs. Four mutations in EO-exposed mice occurred at AT base pairs (two ATCG, two ATTA); these two mutational types did not occur in air controls. Initial inspection of the lacI mutation spectrum determined at 25 and 50 p.p.m. EO revealed no significant differences between the two exposure groups and there was no apparent difference between the lacI mutant or mutation frequencies determined at 25 and 50 p.p.m. (Tables IV and V). Therefore, the DNA sequence data from lacI mutants isolated from the testes at 25 and 50 p.p.m. for 48 weeks was combined for comparison with air controls. A total of 103 lacI mutants isolated from the testes were sequenced from the EO-exposed groups (25 and 50 p.p.m.) and 89 unique mutations were identified, 11% mutant siblings. These groups were selected for sequencing because the lacI mutant frequency was significantly increased at the lowest exposure levels of EO used and these data points determine the initial shape of the exposure–response curve for the lacI mutant frequency in testes (Figure 2a). The purpose of the DNA sequence analysis of testes lacI mutants was to determine the molecular basis for the increased lacI mutant frequency and to determine the contribution of mutant siblings (clonal expansions) to the lacI mutant frequency at the two lowest levels of EO exposure used.
The determination of percentage mutant siblings in the testes samples for each mouse was used to determine the lacI mutation frequency for each exposure group (Table IV), and these data are plotted in Figure 2b. The shape of the exposure–response curve at 0, 25 and 50 p.p.m. EO for the lacI mutation frequency (Figure 2b) was not different from the shape of the exposure–response curve for the lacI mutant frequency (Figure 2a). The lacI mutant frequency in testes determined at the lowest levels of EO exposure was not significantly affected by clonal expansion; only 11% of all lacI mutants analyzed in these groups (25 and 50 p.p.m. EO) were sibling mutations. These data indicate that the increased lacI mutant frequency in testes at the two lowest levels of EO used for 48 weeks was due to the induction of mutations in testes by EO exposure and not due to clonal expansion of specific lacI mutations by EO exposure.
Statistical comparison of the mutation spectrum in testes from air controls compared with EO-exposed mice was done as previously described. Despite the increased mutation frequency in the testes of EO-exposed mice, no specific mutational type was increased to any significant degree. However, there are small but non-significant increases across almost all mutational types. The mutation spectrum analysis of mutations in EO-exposed mice compared with mutations in air control mice isolated from testes did not identify any specific mutational changes in EO-exposed mice. Twelve sites of mutation in testes DNA were in common between air control and EO-exposed mice.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
We determined lacI mutant frequency in the bone marrow and testes of male B6C3F1 lacI transgenic mice exposed by inhalation to EO for up to 48 weeks. Mice were killed at 12, 24 or 48 weeks for the determination of lacI mutant frequency. In the bone marrow of mice, the lacI mutant frequency was increased compared with air controls at the two highest exposure levels (100 and 200 p.p.m.) and only at the 48 week exposure time point. The absence of a detectable mutagenic response at the lacI gene in the bone marrow of mice at the early time points examined (12 and 24 weeks) is consistent with our previous study showing the absence of a mutagenic response at lacI after 4 weeks of exposure to 50, 100 or 200 p.p.m. EO (Sisk et al., 1997
).
At the 48 week time point, EO clearly induced a mutagenic response in the bone marrow (Figure 1). The mutagenic response in bone marrow was non-linear, with no detectable response above air controls at 25 and 50 p.p.m. and an increased lacI mutant frequency above air controls at 100 and 200 p.p.m. EO. The mutational response in the bone marrow at 200 p.p.m. was >3-fold above the air controls and showed a striking mutational specificity. Only a single mutational type was increased among the mutations recovered from the bone marrow, ATTA transversions. This mutational type occurred at a 1.4% frequency in air controls and at a frequency of 25.4% in EO-exposed mice. The increase in mutation frequency and increased frequency of ATTA transversions demonstrates the in vivo induction of point mutations by EO exposure.
In the testes of mice, an increase in the lacI mutant frequency was observed at a single exposure level (200 p.p.m. EO) at 24 weeks. At 48 weeks of EO exposure, the lacI mutant frequency was increased at 25, 50 and 100 p.p.m. EO but not at 200 p.p.m. EO (Figure 2a and b). The lack of an increased lacI mutant frequency at the highest exposure level used and for the longest exposure time (48 weeks) may be due to testicular toxicity of EO (IARC, 1994). Since lacI transgenes are neutral, cytotoxic effects of EO affect cells harboring mutant lacI transgenes to an equal degree as cells with wild-type transgenes. The lacI mutation frequencies in testes at 48 weeks (25 and 50 p.p.m.) were increased 2.3- and 2.5-fold compared with air controls. DNA sequence analysis of testes samples revealed that mutant siblings had little impact on the lacI mutant frequency and did not alter the shape of the exposure–response curve for lacI mutation frequency. The spectrum of point mutations in the testes of EO-exposed mice showed no significant increases in any specific mutational type compared with air controls. The mutational spectrum in testes of EO-exposed mice indicated that there were small increases across most mutational types that were sufficient to produce an overall increase in the lacI mutation frequency, although non-significant for a specific type.
Numerous DNA adducts have been described in the literature resulting from EO exposures. The direct exposure of calf thymus DNA to EO results in numerous DNA adducts, including adducts at adenine (Segarback, 1990; Li et al., 1992). The HO-ethyl-N7-G adduct induced by EO exposure is the most abundant and extensively studied EO-derived DNA adduct, which reaches similar levels in DNA across all tissues examined (Walker et al., 1992; Wu et al., 1999). This same DNA adduct also occurs endogenously as part of the background spectrum of DNA lesions present in cells (Wu et al., 1999). The tissue-specific levels of the HO-ethyl-N7-G adduct do not completely reflect the tissue-specific tumor profile induced by EO exposure in B6C3F1 mice (National Toxicology Program, 1988). Although the genotoxic properties of HO-ethyl-N7-G are uncertain, investigators have proposed that depurination and formation of abasic sites by spontaneous or enzymatic depurination may promote base substitutions, frameshift mutations or large deletions induced by EO exposure (Wu et al., 1999). Although HO-ethyl-N7-G may mediate certain genotoxic effects induced by EO, this adduct likely has a limited role in the induction of point mutations in the bone marrow. However, elevated levels of this adduct in the testes may contribute to the increased lacI mutation frequency and spectrum determined in EO-exposed mice in the present study.
Only limited studies exist on the in vivo formation of DNA adducts other than HO-ethyl-N7-G resulting from inhalation exposures to EO. Low levels of the adenine adduct 3-(2-hydroxyethyl)adenine have been detected in vivo after 4 week EO exposures (Walker et al., 1992). Other adducts at adenine and thymine have been detected following reaction of EO with calf thymus DNA (Segarback et al., 1990; Li et al., 1992). These adducts may be responsible for the increase in ATTA transversions observed in bone marrow at 48 weeks of exposure in the present study. We hypothesize that the difference in mutation spectrum between bone marrow and testes may be due to differential repair of DNA adducts in bone marrow compared with testes.
The mutational spectrum determined in EO-exposed human cells in vitro indicates that large deletions account for 50% of the mutations induced by EO and that point mutations represent the remainder of HPRT mutations (Bastlova et al., 1993). In mice and rats, short-term exposure levels are known to induce a broad range of chromosomal aberrations, micronuclei and other cytogenetic alterations (reviewed in IARC, 1994). In rats, inhaled EO induces an increased frequency of chromosomal alterations and Hprt mutations after a 4 week exposure (Lorenti-Garcia et al., 2001; van Sittert et al., 2000). However, the proportions at which these genotoxic and mutational events occur in vivo are uncertain, particularly under the long-term exposure conditions used here. The absence of point mutations recoverable in the lacI transgene associated with short-term 4 week (Sisk et al., 1997) to 12 week exposures and the increased frequency of chromosomal changes and Hprt mutations with 4 week exposures (Walker et al., 1997) are consistent with the induction of deletions and chromosomal aberrations but not specific point mutations as a primary mechanism of EO-induced genotoxicity or mutagenicity in mice following short-term exposures.
In conclusion, we determined the lacI mutant frequency in two tissues of B6C3F1 lacI transgenic mice exposed to the genotoxic carcinogen EO by inhalation for up to 48 weeks. Under the conditions of EO exposure used in this study, the shape of the exposure–response curves for lacI mutant frequency and the mutational spectrum were different in the two tissues examined. These data demonstrate that inhalation exposure to EO produces distinct mutagenic responses in bone marrow and testes.
|
Supplementary material |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
Supplementary material can be found at: http://www.mutage. oupjournals.org/.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
We thank the CIIT inhalation facility and Mr Arden James for coordinating and monitoring EO exposures. We thank the CIIT animal care facility and necropsy staff. We thank Dr Barbara Kuyper for editorial review. We thank Drs Melvin Anderson, Rory Conolly and David Dorman for their comments. Dr Maria Donner was supported in part by NIH NRSA Grant ES055693-02.
|
Conflict of interest statement |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
This work was done under the direction of L.Recio whilst he was a staff scientist at CIIT; he was paid a consulting fee to write this paper after his separation from CIIT.
|
Notes |
|---|
5To whom correspondence should be addressed. Tel: +1 215 652 5006; Fax: +1 215 652 3888; Email: leslie_recio{at}merck.com
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgmentsConflict of interest statementReferences |
|---|
-
Appelgren,L.E., Eneroth,G., Grant,C., Lanstrom,L.E. and Tenghagen,K. (1978) Testing of ethylene oxide for mutagenicity using the micronucleus test in mice and rats. Acta Pharmacol. Toxicol., 43, 69–71.
Bastlova,T., Andersson,B., Lambert,B. and Kolman,A. (1993) Molecular analysis of ethylene oxide-induced mutations at the HPRT locus in human diploid fibroblasts. Mutat. Res., 287, 283–292.[Web of Science][Medline]
Bird,A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature, 321, 209–213.[CrossRef][Medline]
Callahan,J.D. and Short,J.M. (1995) Transgenic /lacI mutagenicity assay: statistical determination of sample size. Mutat. Res., 327, 201–208.[CrossRef][Web of Science][Medline]
Carr,G.J. and Gorelick,N.J. (1996) Mutational spectra in transgenic animal research: data analysis and study design based upon the mutant or mutation frequency. Environ. Mol. Mutagen., 28, 405–413.[CrossRef][Web of Science][Medline]
de Boer,J.G., Provost,S., Gorelick,N.J., Tindall,K. and Glickman,B.W. (1998) Spontaneous mutation in lacI transgenic mice: a comparison of tissues. Mutagenesis, 13, 109–14.
Dellarco,V.L., Generoso,W.M., Sega,G.A, Fowle,J.R. and Jacobson-Kram,D. (1990) Review of the mutagenicity of ethylene oxide. Environ. Mol. Mutagen., 16, 85–103.[Web of Science][Medline]
Farabaugh,P.J., Schmeissner,U., Hofer,M. and Miller,J.H. (1978) Genetic studies of the lacI repressor. J. Mol. Biol., 126, 847–863.[CrossRef][Web of Science][Medline]
Generoso,W.M., Cain,K.T., Krishna,M., Sheu,C.W. and Gryder,R.M. (1980) Heritable translocation and dominant-lethal mutation induction with ethylene oxide in mice. Mutat. Res., 73, 133–142.[CrossRef][Web of Science][Medline]
Generoso,W.M., Cain,K.T., Hughes,L.A., Sega,G.A., Braden,P.W., Gosslee,D.G. and Shelby,M.D. (1986) Ethylene oxide dose and dose-rate effects in the mouse dominant-lethal test. Environ. Mutagen., 8, 1–7.[Web of Science][Medline]
Generoso,W.M., Cain,K.T., Cornett,C.V., Cachiero,N.L.T. and Hughes,L.A. (1990) Concentration–response curves for ethylene oxide-induced heritable translocations and dominant lethal mutations. Environ. Mol. Mutagen., 16, 126–131.[Web of Science][Medline]
Gossen,J.A., Martus,H.-J., Wei,J.Y. and Vijg,J. (1995) Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model. Mutat. Res., 331, 89–97.[CrossRef][Web of Science][Medline]
13. IARC (1994) Some industrial chemicals. IARC Sci. Publ., 60, 73–159.
Kligerman,A.D., Erexson,G.L., Phelps,M.E. and Wilmer,J.L. (1983) Sister chromatid exchange induction in peripheral blood lymphocytes of rats exposed to ethylene oxide by inhalation. Mutat. Res., 120, 37–44.[CrossRef][Web of Science][Medline]
Kohler,S.W., Provost,G.S., Fieck,A., Kretz,P.L., Bullock,W.O., Sorge,J.A., Putman,D.L. and Short,J.M. (1991) Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc. Natl Acad. Sci. USA, 88, 7958–7962.
Kolman,A., Chovanec,M. and Osterman-Golkar,S. (2002) Genotoxic effects of ethylene oxide, propylene oxide and epichlorohydrin in humans: update review (1990–2001). Mutat Res., 512, 173–194.[CrossRef][Web of Science][Medline]
Lewis,S.E., Barnett,L.B., Felton,C., Johnson,F.M., Skow,L.C., Cacheiro,N. and Shelby,M.D. (1986) Dominant visible and electrophoretically expressed mutations induced in male mice exposed to ethylene oxide by inhalation. Environ. Mutagen., 8, 867–872.[Web of Science][Medline]
Li,F., Segal,A. and Solomon,J.J. (1992) In vitro reaction of ethylene oxide with DNA and characterization of DNA adducts. Chem. Biol. Interact., 83, 35–54.[CrossRef][Web of Science][Medline]
Lorenti-Garcia,C., Darroudi,F., Tates,A.D. and Natarajan,A.T. (2001) Induction and persistence of micronuclei, sister-chromatid exchanges and chromosomal aberrations in splenocytes and bone-marrow cells of rats exposed to ethylene oxide. Mutat. Res., 492, 59–67.[Web of Science][Medline]
Lynch,D.W, Lewis,T.R., Moorman,W.J., Burg,J.R., Gulati,D.K., Kaur,P. and Sabharwal,P.S. (1984) Sister chromatid exchanges and chromosome aberrations in lymphocytes from monkeys exposed to ethylene oxide and propylene oxide by inhalation. Toxicol. Appl. Pharmacol., 76, 85–95.[CrossRef][Web of Science][Medline]
Mirsalis,J.C., Monforte,J.A. and Winegar,R.A. (1994) Transgenic animal models for measuring mutations in vivo. Crit. Rev. Toxicol. 24, 255–280.[Web of Science][Medline]
Natarajan,A.T., Preston,R.J., Dellarco,V., Ehrenberg,L., Generoso,W., Lewis,S. and Tates,A.D. (1995) Ethylene oxide: evaluation of genotoxicity data and an exploratory assessment of genetic risk. Mutat. Res., 330, 55–70.[CrossRef][Web of Science][Medline]
23. National Research Council (1996) Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC.
24. National Toxicology Program (1988) Toxicology and Carcinogenesis Studies of Ethylene Oxide in B6C3F1 Mice, NTP Technical Report 326. US Government Printing Office, Washington, DC.
25. National Toxicology Program (2001) Ninth Annual Report on Carcinogens. National Toxicology Program, Research Triangle Park, NC.
Piegorsch,W.W., Lockhart,A.M., Margolin,B.H., Tindall,K.R., Gorelick,N.J., Short,J.M., Carr,G.J., Thompson,E.D. and Shelby,M.D. (1994) Sources of variability in data from a lacI transgenic mouse mutation assay. Environ. Mol. Mutagen., 23, 17–31.[Web of Science][Medline]
Preston,R.J., Fennell,T.R., Leber,A.P., Sielken,R.L. and Swenberg,J.A. (1996) Reconsideration of the genetic risk assessment from ethylene oxide exposures. Environ. Mol. Mutagen., 26, 189–202.
Provost,G.S. and Short,J.M. (1994) Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice. Proc. Natl Acad. Sci. USA, 91, 6564–6568.
Provost,G.S., Kretz,P.L., Hamner,R.T., Matthews,C.D., Rogers,B.J., Lundberg,K.S., Dycaico,M.J. and Short,J.M. (1993) Transgenic systems for in vivo mutation analysis. Mutat. Res., 288, 133–149.[Web of Science][Medline]
Recio,L. and Meyer,K.G. (1995) Increased frequency of mutations at A:T base pairs in the bone marrow of B6C3F1 IacI mice exposed to 1,3-butadiene. Environ. Mol. Mutagen., 26, 1–8.[Web of Science][Medline]
Recio,L., Pluta,L. and Meyer,K.G. (1998) The in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene. Mutat. Res., 401, 99–110.[Web of Science][Medline]
Rhomberg,L., Dellarco,V.L., Siegel-Scott,C., Dearfield,K.L. and Jacobson-Kram,D. (1990) Quantitative estimation of the genetic risk associated with the induction of heritable translocations at low-dose exposure: ethylene oxide as an example. Environ. Mol. Mutagen., 16, 104–125.[Web of Science][Medline]
Ribiero,L.R., Rabello-Gay,M.N., Salvadori,D.M.F. and Pereira,C.A.B. (1987) Cytogenetic effects of inhaled ethylene oxide in somatic and germ cells of mice. Arch. Toxicol., 59, 332–335.[CrossRef][Web of Science][Medline]
Saranko,C.J., Meyer,K.G., Pluta,L.J., Henderson,R.F. and Recio,L. (2001) Lung-specific mutagenicity and mutational spectrum in B6C3F1 lacI transgenic mice following inhalation exposure to 1,2-epoxybutene. Mutat. Res., 473, 37–49.[Web of Science][Medline]
Segarback,D. (1990) Reaction products in hemoglobin and DNA after in vitro treatment with ethylene oxide and N-(2-hydroxyethyl)-N-nitrosourea. Carcinogenesis, 11, 307–312.
Sisk,S.C., Pluta,L.J., Bond,J.A. and Recio,L. (1994) Molecular analysis of lacI mutants from bone marrow of B6C3F1 transgenic mice following inhalation exposure to 1,3-butadiene. Carcinogenesis, 15, 471–477.
Sisk,S.C., Pluta,L.J., Meyer,K.G., Wong,B.C. and Recio,L. (1997) Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure. Mutat. Res., 391, 153–164.[Web of Science][Medline]
Snellings,W.M., Weil,C.S. and Maronpot,R.R. (1984) A two-year inhalation study of carcinogenic potential of ethylene oxide in Fischer 344 rats. Toxicol. Appl. Pharmacol., 75, 105–117.[CrossRef][Web of Science][Medline]
Tates,A.D., van Dam,F.J., Natarajan,A.T. et al. (1999) Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide. Mutat. Res., 431, 397–415.[Web of Science][Medline]
Their,R. and Bolt,H.M. (2000) Carcinogenicity and genotoxicity of ethylene oxide: new aspects and recent advances. Crit. Rev. Toxicol., 30, 595–608.[Web of Science][Medline]
41. US Environmental Protection Agency (1991) Preliminary draft list of categories and subcategories under Section 112 of the Clean Air Act. Fed. Reg., 56, 28548–28557.
van Sittert,N.J., Boogaard,P.J., Natarajan,A.T., Tates,A.D., Ehrenberg,L.G. and Tornqvist,M.A. (2000) Formation of DNA adducts and induction of mutagenic effects in rats following 4 weeks inhalation exposure to ethylene oxide as a basis for cancer risk assessment. Mutat. Res., 447, 27–48.[Web of Science][Medline]
Walker,V.E. and Skopek,T.R. (1993) A mouse model for the study of in vivo mutational spectra: sequence specificity of ethylene oxide at the hprt locus. Mutat. Res., 288, 151–162.[Web of Science][Medline]
Walker,V.E., Fennell,T.R., Upton,P.B., Skopek,T.R., Prevost,V., Shuker,D.E. and Swenberg,J.A. (1992) Molecular dosimetry of ethylene oxide: formation and persistence of 7-(2-hydroxyethyl)guanine in DNA following repeated exposures of rats and mice. Cancer Res., 52, 4328–4334.
Walker,V.E., Recio,L., Sisk,S.C. and Skopek,T.R. (1997) In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure. Mutat. Res., 392, 211–222.[Web of Science][Medline]
Walker,V.E., Wu,K.Y., Upton,P.B., Ranasinghe,A., Scheller,N., Cho,M.H., Vergnes,J.S., Skopek,T.R. and Swenberg,J.A. (2000) Biomarkers of exposure and effect as indicators of potential carcinogenic risk arising from in vivo metabolism of ethylene to ethylene oxide. Carcinogenesis, 21, 1661–1669.
Wu,K.Y., Ranasinghe,A., Upton,P.B., Walker,V.E. and Swenberg,J.A. (1999) Molecular dosimetry of endogenous and ethylene oxide-induced N7-(2-hydroxyethyl) guanine formation in tissues of rodents. Carcinogenesis, 20, 1787–1792.
Yager,J.W. and Benz,R.D. (1982) Sister chromatid exchanges induced in rabbit lymphocytes by ethylene oxide after inhalation exposure. Environ. Mutagen., 4, 121–134.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. M. Donner, B. A. Wong, R. A. James, and R. J. Preston Reciprocal translocations in somatic and germ cells of mice chronically exposed by inhalation to ethylene oxide: implications for risk assessment Mutagenesis, November 1, 2009; (2009) gep042v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-H. L. Hong, C. D. Houle, T.-V. T. Ton, and R. C. Sills K-ras Mutations in Lung Tumors and Tumors from Other Organs are Consistent with a Common Mechanism of Ethylene Oxide Tumorigenesis in the B6C3F1 Mouse Toxicol Pathol, January 1, 2007; 35(1): 81 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Houle, T.-V. T. Ton, N. Clayton, J. Huff, H.-H. L. Hong, and R. C. Sills Frequent p53 and H-ras Mutations in Benzene- and Ethylene Oxide-Induced Mammary Gland Carcinomas from B6C3F1 Mice Toxicol Pathol, October 1, 2006; 34(6): 752 - 762. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||