In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure

Mutation Research 392 Ž1997. 211–222

In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure Vernon E. Walker

a,b,)

, Susan C. Sisk c , Patricia B. Upton d , Brian A. Wong c , Leslie Recio c

a

Department of Pathology, UniÕersity of North Carolina at Chapel Hill, CB No. 7525, Chapel Hill, NC 27599-7525, USA Wadsworth Center for Laboratories and Research, NY State Department of Health, P.O. Box 509, Albany, NY 12201-0509, USA c Chemical Industry Institute of Toxicology, 6 DaÕis DriÕe, Research Triangle Park, NC 27709, USA Department of EnÕironmental Sciences and Engineering, UniÕersity of North Carolina at Chapel Hill, CB No. 7525, Chapel Hill, NC 27599-7525, USA b

d

Received 7 October 1996; revised 24 January 1997; accepted 12 February 1997

Abstract Ethylene oxide ŽEO. is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase Ž hprt . locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue w mice at 6–8 Ž n s 4rgroup. and 8–10 Ž n s 5rgroup. weeks of age were exposed to 0, 50, 100, or 200 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek.. At necropsy, T-cells were isolated from thymus andror spleen and cultured in the presence of concanavalin A, IL-2, and 6-thioguanine wSkopek, T.R., V.E. Walker, J.E. Cochrane et al. Ž1992. Proc. Natl. Acad. Sci. USA, 89, 7866–7870x. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency ŽM f . in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5 " 0.9 = 10y6 Žaverage M f " SE. at 2 weeks post-exposure, compared with 2.3 " 0.8 = 10y6 in thymus of control mice. Dose-related increases in hprt M f s were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt M f s was observed in splenic T-cells, with greater mutagenic efficiency Žmutations per unit dose. found at higher concentrations than at lower concentrations of EO. Average induced M f s Ži.e. induced M f s treatment M f y background M f . in splenic T-cells were 1.6, 4.6, and 11.9 = 10y6 following exposures to 50, 100, or 200 ppm EO, respectively, while the average control M f value was 2.2 " 0.3 = 10y6 . In aliquots of lymphocytes Žboth B- and T-cells. isolated from spleen for analysis of lacI mutations in the same animals, only two of three

Abbreviations: AP sites, abasic sites; ENU, ethylnitrosourea; EO, ethylene oxide; N7-HEG, N 7-Ž2-hydroxyethyl.guanine; hprt, hypoxanthine guanine phosphoribosyltransferase; IL-2, interleukin-2; M f, mutant frequency ) Corresponding author, at address b. Tel.: q1 Ž518. 474-4046; Fax: q1 Ž518. 486-1505; e-mail: [email protected] 1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 6 2 - 4

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EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI M f and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure wSisk, S., L.J. Pluta, K.G. Meyer and L. Recio Ž1996. Mutation Res., submittedx. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin. q 1997 Elsevier Science B.V. Keywords: Dose response; Ethylene oxide; Hprt; In vivo mutation; LacI; Transgenic mice

1. Introduction Ethylene oxide ŽEO; CAS a75-21-8., a major chemical intermediate and gaseous sterilant, is a direct-acting alkylating agent that is genotoxic and carcinogenic in several species w3–5x. Two-year inhalation studies using exposure concentrations in the range of 10–100 ppm EO have shown that this agent produces increased incidences of several tumor types in F344 rats w6x and B6C3F1 mice w7x, including T-cell lymphomas in female mice. Exposure to relatively high concentrations of EO has also been suggested as a cause of non-Hodgkin’s lymphoma in occupationally exposed workers, based upon epidemiological data w8x; while exposure of people to similarly high or much lower concentrations of EO has been associated with increased frequencies of several markers of exposure and effect, including hemoglobin adducts, sister-chromatid exchanges, micronuclei, and chromosomal aberrations w9–12x. Based partially upon these data, the International Agency for Research on Cancer recently classified EO as a Group 1 carcinogen Ži.e. carcinogenic in humans based on ‘‘sufficient evidence in animals together with strong evidence in humans of a relevant mechanism for carcinogenicity’’. w5x. The induction of mutation and initiation of cancer by alkylating agents, such as EO, are thought to be the result of damage produced at critical sites in DNA w13x. EO acts by the S N 2 mechanism w14x, and its reaction with DNA in vitro leads mainly to the formation of N 7-Ž2-hydroxyethyl.guanine ŽN7HEG., N 3-Ž2-hydroxyethyl.adenine, and lesser amounts of several other DNA adducts w15,16x. NAlkylation of DNA bases, with subsequent production of non-coding abasic sites ŽAP sites; apurinic sites. by spontaneous or enzymatic depurination, may contribute to EO-induced mutagenesis w14x. The rela-

tionships between in vivo exposure and DNA adduct formation and persistence have been thoroughly investigated in mice and rats following single w17,18x and multiple exposures w4,19,20x to EO; however, no significant correlations have been found between DNA adduct concentrations and tissue specificity for EO-induced carcinogenesis w21x. Nevertheless, an examination of the types of mutations induced by EO should indicate which types of DNA adductŽs. are causally related to the mutagenic mechanisms of this agent w14,19,21x. The mutagenic potency and specificity of EO have been recently evaluated at the hypoxanthine guanine phosphoribosyltransferase Ž hprt . locus in human diploid fibroblasts cultured in vitro w22,23x and in T-lymphocytes from preweanling B6C3F1 mice exposed in vivo by i.p. injections w14x. In both systems, exposures to high concentrations of EO produced dose dependent increases in the frequency of hprt mutations. Molecular characterization of EO-induced mutations in human fibroblasts in vitro suggested that large deletions are a prominent class of mutations caused by this agent at hprt w23x, while the limited in vivo mutational spectra data in mice indicated that modifications at both guanine and adenine bases are involved in EO mutagenesis w14x. The purpose of the present study was to determine the effects of EO exposure level and time elapsed after exposures on the frequency of hprt-negative lymphocytes from B6C3F1 lacI transgenic mice exposed by inhalation. Since workers are most commonly exposed to EO in the gaseous form, inhalation exposure is a more appropriate route of administration for evaluating the relationships between EO exposure and the induction of mutations in experimental animals. This study was part of a larger collaborative effort to evaluate the relative sensitivities and specificities of the endogenous hprt gene

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and the lacI transgene as mutational targets in lymphocytes from spleen of B6C3F1 Big Blue w mice exposed to EO w2x.

2. Materials and methods 2.1. Chemicals and media components EO Žpurity 99.99%. was obtained from SunOx Corp. Lympholyte w M was purchased from Accurate Chemical and Scientific Corp. ŽWestbury, NY.. Medium components for hprt mutation assays were obtained from the indicated sources: fetal bovine serum, RPMI 1640 medium, Hepes buffer, glutamine, MEM non-essential amino acids, pen-strep, and sodium pyruvate ŽGibco Laboratories.; HL-1 medium ŽVentrex Laboratories, Portland, ME.; and 2-mercaptoethanol, concanavalin A, recombinant mouse interleukin-2 ŽIL-2., and 6-thioguanine ŽSigma.. Lymphokine activated killer T-cell treatment supernatant containing human IL-2 was a gift from Dr. Timothy Darrow ŽDuke University.. 2.2. Animals, husbandry and exposures Groups of male B6C3F1 Big Blue w Ž lacI transgenic. mice at 4–6 and 6–8 weeks of age were obtained from Stratagene Cloning Systems, Inc. ŽTaconic Farms, Germantown, NY.. All animals were free of virus titers, as determined by standard mouse virus antibody assays ŽMicrobiological Associates, Bethesda, MD.. The housing of animals conformed with NIH guidelines ŽNIH Publication no. 86-23, 1985.. Animals were housed in temperature- and humidity-controlled rooms Ž72 " 48F and 50 " 10%. with a 12:12-h lightrdark cycle in polystyrene cages containing heat-sterilized wood chips. The mice were acclimated for two weeks prior to treatment, and had free access to food ŽNIH-07 certified feed. and water at all times except during 6 h periods of EO exposure. Mice were weighed weekly throughout the animal study. Animal exposures were whole-body, with concentrations of EO in exposure chambers monitored at least once an hour using a Hewlett-Packard 5890 Series II gas chromatograph equipped with a flame ionization detector. The average inhalation chamber

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concentrations for the 4-week exposure periods were 51.9 " 1.6, 102.4 " 2.5, and 196.5 " 4.3 ppm Žaverage " SD. EO. Control mice were exposed to chamber supply air with 0 ppm EO in a sham exposure. During exposures, mice were housed individually in hanging steel cages inside a 1-m3 Hinners-style chamber and deprived of food and water. EO-treated and control lacI transgenic mice were euthanized at specified times post-exposure by CO 2 asphyxiation followed by exsanguination by cardiac puncture, and their thymus andror spleens were removed aseptically for isolation of T-lymphocytes. At the same time, additional untreated nontransgenic B6C3F1 mice were necropsied to provide a source of splenic lymphocytes to be used as syngeneic ‘feeder cells’. Several overlapping experiments were conducted using lacI transgenic mice Ži. to characterize the effect of EO exposure and time elapsed after EO administration on the frequency of hprt mutations in T-cells from thymus, Žii. to determine the relationship between EO concentration and hprt M f s in T-cells from thymus and spleen, and Žiii. to collect hprt mutant T-cell colonies for molecular analysis. Each treatment group contained five mice at 8–10 weeks of age at the beginning of exposures. To define the time course for the appearance of hprt mutations in T-cells of thymus from exposed mice, groups of animals were exposed to 0 or 200 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek. and necropsied 2 h and 2 and 8 weeks post-exposure for collection of thymus and measurement of M f s. To determine the dose-response for hprt mutant T-cells, groups of mice were exposed to 0, 50, 100 or 200 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek. and necropsied 2 weeks after treatment to collect thymus and 8 weeks post-exposure for collection of spleen and measurement of M f s. To compare the endogenous hprt gene and lacI transgene as mutational targets, groups Ž n s 4rgroup. of 6–8-week-old male Big Blue mice were exposed concurrently to 0 to 200 ppm EO for 4 weeks and necropsied 8 weeks post-exposure for collection of spleen and determination of M f s at both loci within the same animals w2x. The use of two different age groups in these experiments Ž6- to 8-week-old primarily for lacI investigations and 8to 10-week-old primarily for hprt investigations.

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was necessitated by the ages of the animals available for the overall EO study. 2.3. Isolation of lymphocytes from spleen and thymus The procedure used for isolating lymphocytes from spleen and thymus has been described in detail previously w1,14x. Briefly, lymphocytes were isolated by macerating spleens and thymus individually in medium, washing and suspending the cells in medium, and layering the cells on a ficoll gradient ŽLympholyte M. for collection, washing, and resuspension in supplemented medium. Examination of Wright-stained smears of cell pellets from spleen demonstrated that this process results in cell isolations containing approximately 98% lymphocytes Žboth B- and T-cells. and 2% neutrophils. Following isolation of lymphocytes from control and EO-treated lacI transgenic mice, the cells were enumerated using a Coulter Counter and adjusted to 1 = 10 6 cellsrml medium. As part of a collaborative study between coauthors of this report, 50% of the suspended cells from each spleen of mice exposed at 6–8 weeks of age were used for hprt mutation assays, while the remaining cells were centrifuged to collect the cell pellet for extraction of DNA and determination of lacI M f s in splenic lymphocytes ŽB- and T-cells. of the same animals w2x. All suspended cells from spleens of mice treated at 8–10 weeks of age and every thymus were used for growth and selection of hprt mutant T-cells. 2.4. Isolation of hprt mutant lymphocytes The isolation of hprt mutant lymphocytes has been described previously w1,14x. Briefly, T-cells were cultured in RPMI 1640 medium supplemented with mitogen Žconcanavalin A., growth factor ŽIL-2. and selective agent Ž6-thioguanine. in 96-well microtiter plates. To determine the plating efficiency for lymphocytes from each lacI transgenic mouse, eight mouse cellsrwell were cultured in the presence of lethally-irradiated splenic lymphocytes Ž‘feeder’ cells. isolated from untreated nontransgenic B6C3F1 mice. To isolate hprt mutants, cells that had been incubated in T-flasks for 24 h were counted and diluted to 4 = 10 5 cellsrml for seeding into 96-well microtiter plates at 40 000 cellsrwell for incubation.

Plates were scored at 4 = magnification on day 12. Hprt M f was calculated as described previously w1x, with M f data for individual treatment groups expressed as the average M f " standard error of the mean ŽSE.. Hprt mutant clones from spleens of control and EO-exposed mice were collected and frozen for future molecular analyses. 2.5. Statistical analyses The Mann–Whitney Test Žunpaired, two-tailed. was used to evaluate the statistical difference between the hprt M f data from the controls and groups of EO-exposed lacI transgenic mice. A pvalue - 0.05 was considered significant.

3. Results EO exposures up to 200 ppm for 4 weeks caused no clinical signs of toxicity in lacI transgenic mice, and the average body weight gain of the treatment groups was not significantly different from that of the control groups w2x. The plating efficiencies of lymphocytes isolated from thymus Ž0.6–3.9%. and spleen Ž1.6–4.2%. of treated mice were similar to those of control animals Žthymus: 1.5–3.6%; spleen: 2.4–4.1%.. The average hprt mutant fractions in T-cells from control mice were 2.2 " 0.8 = 10y6 Žaverage " SE. clonable cells in thymus and 2.2 " 0.3 = 10y6 in spleen. These cloning efficiency and M f values resemble those reported previously for lymphocytes from thymus and spleen of untreated male and female nontransgenic and lacI transgenic B6C3F1 mice w1,14,24–26x. The first experiment in this study assessed the effect of EO treatment on the time course for the appearance of hprt M f s in T-cells from thymus following 4 weeks of exposure of lacI transgenic mice to 200 ppm EO ŽFig. 1.. Significantly elevated M f s were apparent in treated animals necropsied immediately after exposure Žaverages 5.8 " 0.6 = 10y6 ; p s 0.018., and M f s continued to increase in exposed mice through at least 2 weeks post-exposure Žaverages 7.5 " 0.9 = 10y6 ; p s 0.018. before declining to lower values approaching background in some animals at 8 weeks after exposure. A similar pattern of mutant expression Ži.e. phenotypic expres-

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Fig. 1. Relationship between the time elapsed since the cessation of ethylene oxide ŽEO. exposure and the frequency of hprt mutant lymphocytes from thymus of exposed male B6C3F1 lacI transgenic mice. Groups of mice Ž8–10 weeks of age. were exposed to 0 Ž`. or 200 Žv . ppm EO for 4 weeks Ž6 hrday, 5 daysrweek. by inhalation, and then thymus were collected for T-cell isolation and culture at 0, 2 and 8 weeks after the last exposure. Points, averages; bars, SE Ž ns 5rgroup, except for 3rgroup at 8 weeks post-exposure.; average control hprt mutant frequency value of 2.3"0.8=10y6 Ž ns 5rgroup..

sion of mutant hprt protein and T-cell migration. has been observed in T-cells from thymus of generic B6C3F1 mice of similar age and sex following in vivo exposures to ethylnitrosourea w27x. The dose-response for hprt mutant lymphocytes in thymus was assessed in mice necropsied 2 weeks after exposure to 0, 50, 100, or 200 ppm EO for 4 weeks ŽFig. 2.. Hprt M f s in thymus from animals exposed to 50 ppm EO were not different from those in control mice. However, repeated exposures to higher concentrations led to significant dose-related increases in M f s that were 1.7- Ž p s 0.036. and 3.3-fold Ž p s 0.018. above background in the 100 and 200 ppm EO treatment groups, respectively. The dose-response for hprt mutant T-cells in spleen was evaluated in mice necropsied 8 weeks after exposure to 0 to 200 ppm EO for 4 weeks ŽTable 1.. EO exposures led to significant dose-related increases in observed M f s that were 1.7-, 3.1-, and 6.4-fold above background Ž2.2 " 0.3 = 10y6 . in the 50, 100, and 200 ppm treatment groups, respectively. The variation in the age of animals at the beginning of EO exposure Ž6–8 versus 8–10

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weeks of age. did not cause a significant difference in the mutagenic response in splenic T-cells. A cursory inspection of the observed treatment M f values might suggest that EO produced a linear dose-response for hprt mutant T-cells in spleen ŽTable 1; Fig. 3A.; however, a careful examination using the average induced M f s Ži.e. induced M f s observed treatment M f y background M f . in splenic T-cells indicates that repeated exposures to EO actually resulted in a sublinear dose-response, with greater mutagenic efficiency found at higher exposure concentrations than at lower concentrations ŽTable 1.. For example, the average induced M f s at 100 and 200 ppm EO Ži.e. 4.6 and 11.9 = 10y6 , respectively. were 44% and 86% greater, respectively, than the values predicted by that observed at 50 ppm EO Ži.e. 1.6 = 10y6 .. Furthermore, the ratio of induced hprt M f to exposure concentration, an index of the efficiency of mutant induction, increased from 3.2 to 6.0 = 10y8 as the exposure concentration was increased from 50 to 200 ppm EO ŽFig. 3B.. If the dose–response curve for hprt mutations were linear, plotting of these ratios Žinduced M frexposure concentration. against the exposure concentration would have yielded a horizontal line in Fig. 3B.

Fig. 2. Dose-response for hprt mutant lymphocytes from thymus of exposed male B6C3F1 lacI trangenic mice following repeated inhalation exposures to ethylene oxide ŽEO.. Groups of mice Ž8–10 weeks old. were exposed to 0, 50, 100 or 200 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek., and then thymus were collected for T-cell isolation and culture at 2 weeks after the last exposure. Points, averages; bars, SE Ž ns 5rgroup..

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Table 1 hprt Mutant frequencies in T-lymphocytes from spleen of male Big Blue w B6C3F1 mice following inhalation exposures to ethylene oxide Exposure Žppm.

Animal no.

0

201 a 204 a 11 12 15

50

100

200

a

206 208 a 26 27 28 29 30 209 a 211 a 212 a 41 42 43 44 45 213 a 214 a 215 a 216 a 56 57 58 59 60

Observed mutant frequency Ž=10y6 .

Average induced mutant frequency Ž=10y6 .

b

P value

c

2.1 2.8 1.6 1.8 2.6 Mean " SE, 2.2 " 0.3

0

–

Mean " SE, 3.8 " 0.5

1.6

0.009

Mean " SE, 6.8 " 0.9

4.6

0.001

Mean " SE, 14.1 " 1.1

11.9

- 0.001

5.1 5.4 3.2 2.5 2.7 3.4 4.5 5.6 7.0 11.5 3.6 6.6 5.9 9.0 5.5 12.3 12.0 14.0 9.3 19.0 19.6 11.5 14.7 14.8

Groups of mice were 6–8 a and 8–10 weeks old when exposed to ethylene oxide for 4 weeks Ž6 hrday, 5 daysrweek.. Spleens were collected for T-cell isolation and culture at 8 weeks post-exposure. a Spleen cell isolates were divided and evaluated for mutant frequencies at the hprt and lacI loci. b Average induced mutant frequencys Žaverage treatment mutant frequency. y Žaverage background mutant frequency.. c Mann–Whitney, unpaired, two-tailed test.

Based upon the M f values alone, there were no significant outliers within any treatment group that might represent the occurrence of an in vivo clonal amplification of T-cells carrying the same hprt mutation in a given animal. Following DNA sequencing, hprt mutants with identical mutations in T-cell clones isolated from a single animal may represent mutant siblings that arose from a single mutational

event. Therefore, the possibility that mutant T-cell siblings may have contributed to the in vivo hprt mutant frequencies observed in these mice will be considered and M f s reassessed when DNA from mutant colonies is sequenced to define the location and types of mutations induced following inhalation exposures to EO. However, the validity of the hprt M f numbers obtained for control and treated ani-

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gene copies per cell w40x and the higher background M f at the lacI transgene Ž; 10-fold higher than hprt ., the lacI system is actually 400-times Ž40 = 10. more likely to display a clonal expansion than single-copy hprt Ži.e. among all cells undergoing clonal expansion, the probability that one will be carrying a lacI mutation is 400-times the probability that one will be carrying a hprt mutation.. The lacI M f data for EO-exposed mice are discussed in detail in the preceding paper w2x.

4. Discussion

Fig. 3. Comparison of the dose-response for observed frequency of hprt mutant T-cells ŽM f . ŽA. and the efficiency of mutant induction ŽB. in lymphocytes from spleen of exposed male B6C3F1 lacI trangenic mice following repeated inhalation exposures to ethylene oxide ŽEO.. Groups of mice were 6–8 Ž ns 3–4rgroup. and 8–10 Ž ns 5rgroup. weeks old when exposed to 0, 50, 100 or 200 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek.. Spleens were collected for T-cell isolation and culture at 8 weeks after the last EO exposure. In B, the average induced frequency of hprt mutant T-cells Žinduced M f s treatment M f ybackground M f . was divided by the exposure concentration of EO Žppm. and plotted against the exposure concentration. Points, averages; bars, SE.

mals is supported by the fact that similar responses Ži.e. the magnitude and shape of the dose–response curves. were observed in two tissues of EO-exposed mice. In contrast, two of the mice exposed to 200 ppm EO had elevated lacI M f s that were likely due to the in vivo replication of preexisting mutant lymphocytes and not due to the induction of new mutations in these animals. Due to the number of trans-

Experiments were conducted to determine the effects of EO exposure level and time elapsed after inhalation exposures on the frequency of hprt mutant lymphocytes in male B6C3F1 lacI transgenic mice. The design of these experiments was drawn in part from earlier studies with EO. The B6C3F1 mouse was selected as a model for this research because tumor data are available for this strain from the National Toxicology Program cancer bioassay of EO w7x, and subsequently the same strain was used in the development of the mouse T-cell cloningrsequencing assay for hprt mutations w14x. The exposure levels for this project were based upon those used in the carcinogenicity bioassay for EO in mice w7x, while the duration of exposures was based upon earlier studies demonstrating that the major DNA adduct of EO, N7-HEG, approaches steady-state concentrations in target and nontarget tissues of rodents by 4 weeks of exposure w4,19x. The experiment evaluating the time course for mutant appearance in lacI transgenic mice demonstrated that EO produces time-dependent increases in the frequency of hprt-negative lymphocytes in thymus, with the highest average induced M f observed 2 weeks after the last exposure. Although the time period required to reach maximum M f s in thymic T-cells following EO exposure cannot be determined definitively in this experiment owing to the limited number of time points evaluated Ži.e. 0, 2, and 8 weeks post-exposure., a similar pattern of hprt mutant expression Ži.e. both phenotypic expression of mutant hprt protein and T-cell migration. has been observed in T-cells from thymus of male nontransgenic B6C3F1 and C57Bl_ 6 mice of various ages

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following in vivo exposures to ethylnitrosourea ŽENU.. In thymus of mice given single i.p. injections of ENU Ž40 or 58 mgrkg. at 2, 3, 7, or 13 weeks of age, the maximum M f s were found 2 weeks post-exposure in all age groups following isolation of thymic T-cells at necropsies performed weekly or biweekly up to 8 weeks after treatment w27x. Likewise, a similar expression time of 2 weeks was required to achieve maximum M f s in thymic T-cells from groups of female generic B6C3F1 mice Ž4 weeks old. necropsied at weekly intervals for up to 10 weeks after 2 weeks of inhalation exposures to 1250 ppm 1,3-butadiene w26x. These experimental data combined suggest that the time required to reach maximum induced frequencies of hprt mutant T-cells in mouse thymus following chemical exposure is independent of age, sex, and strain of mouse, and that the average hprt M f s shown in Fig. 1 and Fig. 2 are likely to represent the maximum values that could be observed in thymus of EO-treated mice under these exposure conditions Ži.e. 4 weeks at 0 to 200 ppm EO.. Issues concerning the relationships between exposure to simple alkylating agents, animal age at the time of exposure, the expression of hprt mutations in thymus, and T-cell migration will be discussed in detail in a separate paper. The selection of a mutant expression time of 8 weeks post-exposure for evaluating the dose-response for hprt M f s in splenic T-cells of Big Blue mice was based upon the needs of collaborators performing the lacI mutation studies in the same animals w2x and the results of previous studies in ENU-treated nontransgenic mice. The ENU experiments in mice given single doses at 2 to 13 weeks of age demonstrated that the length of time required to reach the maximum frequency of hprt mutant T-cells in spleen following treatment correlated directly with the age of the animal at the time of treatment w27x. Based upon these earlier ENU studies and the ages of the lacI transgenic mice at the end of the EO inhalation studies Ži.e. 10 to 14 weeks old., the maximum hprt M f in splenic T-cells would be predicted to occur 8–10 weeks after the cessation of EO exposures. Nevertheless, it is important to note that studies in mice and rats suggest that the relationship between the time elapsed since chemical exposure and the frequency of hprt mutant T-cells in peripheral lymphoid tissues Ži.e. peripheral circula-

tion, lymph nodes, spleen, and blood rich tissues other than thymus. depends upon the treatment effects of specific alkylating agents as well as age and species related differences in the kinetics of the lymphoid system w27–29x. Exposure of Big Blue mice with EO for 4 weeks resulted in a nonlinear dose-response for induced hprt M f s in T-cells from spleen of animals necropsied 8 weeks after exposure to 50, 100, or 200 ppm. Similar nonlinear dose–response curves for the formation of the major DNA adduct of EO, N7-HEG, have been found following inhalation exposures of male B6C3F1 mice Ž9 weeks old. to 0, 3, 10, 33, or 100 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek. w19,21x. The dose–response relationships for N7HEG were sublinear w30x in the three tissues evaluated, which included brain, lung, and spleen. N7HEG concentrations were not measured specifically in lymphocytes from thymus or spleen, but Fig. 4 compares the shape of the dose–response curve for N7-HEG in whole spleen DNA to the dose–response curve for hprt M f s in splenic T-cells. The dose–re-

Fig. 4. Comparison of the dose-response for 7-Ž2-hydroxyethyl.guanine in DNA of whole spleen Žv . and for induced hprt mutant frequencies ŽM f s. in splenic T-cells Ž`. from male B6C3F1 mice following repeated inhalation exposures to ethylene oxide ŽEO.. Groups of conventional mice were exposed to 0, 10, 33 or 100 ppm EO for 4 weeks Ž6 hrday, 5 daysrweek., and then spleens were collected 2 h after the last exposure to measure DNA adducts Ždata from Ref. w19x.. Groups of transgenic mice were similarly exposed to 0, 50, 100 or 200 ppm EO for 4 weeks, and then spleens were collected for T-cell isolation and culture at 8 weeks after the last exposure to determine induced hprt M f s Ži.e. induced M f s treatment M f ybackground M f ..

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sponse curves for N7-HEG and hprt M f s have the same sublinear shape, indicating that the alkylation and mutagenic efficiencies of EO are greater at higher exposure concentrations associated with the induction of cancers in the mouse w7x. On the other hand, DNA repair of N7-HEG appears to be saturated at these high exposure concentrations w19x, suggesting at first glance that a sublinear DNA adduct curve and a corresponding supralinear mutation curve would be expected if AP sites resulting from repair or spontaneous depurination of N7-HEG are involved in EO mutagenesis. However, the observation of sublinear adduct and mutation response curves can be explained if only AP sites produced from chemical depurinations are involved in EO mutagenesis, since the frequency of these AP sites will be proportional to the number of adducts present in DNA. This possibility is discussed further in a related paper concerning the types of mutations produced by EO and the nature of the promutagenic DNA lesions that may lead to EO-induced mutations w31x. To compare the mutagenic response at the native hprt gene and the lacI transgene of EO-exposed animals, M f s at both loci were measured using the same mononuclear cell fractions isolated from spleens of control and treated mice. While EO produced dose-related increases in hprt M f s in T-cells from spleens of exposed Big Blue mice, the average lacI M f s in splenic mononuclear cell isolates Ži.e. B- and T-cells. from the 50 and 100 ppm EO groups were not significantly different from the air control group w2x. Two of three animals exposed to 200 ppm EO had elevated lacI M f s that resulted in a 4-fold increase over the average background value; however, molecular analysis of the lacI mutants, previous data on the location and types of spontaneous mutations, and statistical evaluations suggested that these M f elevations were not treatment induced but were likely due to the clonal expansion of background mutations during or following the period of EO exposure. On the other hand, the lacI M f in lung was significantly increased at 8 weeks after repeated exposures to 200 ppm EO. Lung is a target tissue for EO-induced cancer in both male and female mice w7x. Details of the lacI analyses and the interpretation of the resulting data are presented in the preceding paper w2x.

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The lack of a detectable mutational response to EO at the lacI locus in lymphocytes from spleen is likely related to a combination of factors, including certain inherent characteristics of the Big Blue system, the mechanisms involved in EO-induced mutagenesis, and the molecular dose of EO delivered to these cells or their precursors. Since these factors have been considered in depth by Sisk et al. w2x, they will only be discussed in brief here. First, the difference in the mutagenic response at the lacI and hprt loci in spleen cells of EO-treated animals may be due in part to a variation in the ability of each mutation system to detect large-scale mutations. Current information suggests that the Big Blue system detects primarily base substitutions and small-scale deletions and insertions, while large-scale deletions are not readily detected w32–34x. The hprt assay detects both small- and large-scale mutations Žspecifically, deletions up to a megabase have been recorded at hprt .. This difference is important because several lines of evidence suggest that EO is a potent clastogen and may produce a high frequency of large deletions that could contribute to an increased M f at the hprt locus w3,23,35,36x. Finally, the high baseline for background mutations in lacI may make it more difficult to resolve spontaneous and induced M f s and to demonstrate clearly the mutagenic effects of some alkylating agents in lacI compared to hprt w25,37,38x. For example, in the present study the average M f in lacI of splenic lymphocytes from untreated lacI transgenic mice was 30 " 12 = 10y6 w2x compared with an average background M f of 2.2 " 0.3 = 10y6 in hprt of splenic T-cells from the same animals, while the induced M f s at hprt in the high exposure Ž200 ppm. EO group was 11.9 = 10y6 ŽTable 1.. A comparison of the average induced hprt M f of 11.9 = 10y6 with the lacI background M f of 30 = 10y6 , considering the typical interanimal variability in lacI M f Ži.e. "12 = 10y6 in this study., illustrates that induced M f s similar to those observed in hprt under these exposure and sampling conditions are likely too small to be detectable in the transgene. In addition to the present studies Žw2x; present paper., several other investigations have been undertaken to compare the quantitative and qualitative nature of mutations occurring at the hprt gene and lacI transgene of mutagen-treated mice. Other model

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compounds being investigated include ethylnitrosourea as a model S N 1 direct-acting alkylating agent w25x, and benzow axpyrene w39x and cyclophosphamide w40x as model indirect-acting alkylating agents. A brief comparison of the preliminary results of these investigations have been presented elsewhere w40x. The current study demonstrates that repeated inhalation exposures to G 50 ppm EO produce doserelated increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin. However, interpretations of these results are complicated by the fact that, in the carcinogenicity bioassay of EO in B6C3F1 mice, the incidence of malignant lymphomas in males was dose-related but not significantly increased, while the incidence of this malignancy in females was significantly increased at the high dose Ž100 ppm EO. but not the low dose Ž50 ppm EO. w7x. Because exposure to relatively high concentrations of EO has been linked to non-Hodgkin’s lymphoma in occupationally exposed workers and produces an elevated incidence of thymic lymphomas in female mice, the relevance of the mouse lymphoma model for risk extrapolation needs to be investigated by determining associations between EO exposures, induction of T-cell mutations, and incidence of lymphomas in female mice specifically. Nevertheless, the Žhemoglobin and DNA. adduct data w20,21x and hprt M f data obtained in similarly exposed male mice provide a means of examining relationships between external exposures to EO, blood concentrations of EO, internal dose to DNA, and frequency of induced mutations at an expressed marker gene. Furthermore, molecular characterization of EO-induced mutations in mutant T-cells collected from male and female nontransgenic w14,31x and male transgenic Žpresent study. B6C3F1 mice may indicate the nature of the DNA adducts leading to these mutations.

Acknowledgements This work was supported, in part, by grants from the Ethylene Oxide Industry Council ŽChemical Manufacturer’s Association. and the National Cancer

Institute Žgrant no. IF32 CA612722-01.. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. Thanks are due to Susan C.J. Sumner for her role in discussions leading to this collaboration, to James A. Swenberg and Thomas R. Skopek for valuable discussions and help in the soliciting support for this work, to the CIIT inhalation facility scientists and staff for EO exposures, and to Timothy R. Fennell and Julian R. Preston for a critical reading of the manuscript.

References w1x T.R. Skopek, V.E. Walker, J.E. Cochrane, T.R. Craft, N.F. Cariello, Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea, Proc. Natl. Acad. Sci. USA 89 Ž1992. 7866–7870. w2x Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio Ž1996. Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure, Mutation Res., submitted. w3x Environmental Protection Agency ŽEPA. Ž1990. Office of Health and Environmental Assessment, V.L. Dellarco ŽEd.., U.S. EPA Genetic risk assessment on ethylene oxide, Environ. Mol. Mutagen., 16, 81–134. w4x V.E. Walker, T.R. Fennell, J.A. Boucheron, N. Fedtke, F. Ciroussel, J.A. Swenberg, Macromolecular adducts of ethylene oxide: a literature review and a time-course study on the formation of 7-Ž2-hydroxyethyl.guanine following exposures of rats by inhalation, Mutation Res. 233 Ž1990. 151–164. w5x International Agency for Research on Cancer ŽIARC. Ž1994. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Some Industrial Chemicals, Vol. 60., IARC Sci. Publ., Lyon. w6x W.M. Snellings, C.S. Weil, R.R. Maronpot, A two-year inhalation study of carcinogenic potential of ethylene oxide in Fischer 344 rats, Toxicol. Appl. Pharmacol. 75 Ž1984. 105–117. w7x National Toxicology Program ŽNTP., Department of Health and Human Services Ž1988. Toxicology and carcinogenesis studies of ethylene oxide in B6C3F1 mice Žinhalation studies., NTP Technical Report, Vol. 326, USDHHS, U.S. Government Printing Office, Washington, DC. w8x R.E. Shore, M.J. Gardner, B. Pannett, Ethylene oxide: an assessment of the epidemiological evidence on carcinogenicity, Br. J. Ind. Med. 50 Ž1993. 971–997. w9x J. Mayer, D. Warburton, A.M. Jeffrey, R. Pero, S. Walles, L. Andrews, M. Toor, L. Latriano, L. Wazneh, D. Tang, W.-Y. Tsai, M. Kuroda, F. Perera, Biologic markers in ethylene oxide-exposed workers and controls, Mutation Res. 248 Ž1991. 163–176.

V.E. Walker et al.r Mutation Research 392 (1997) 211–222 w10x A.D. Tates, T. Grummt, M. Tornqvist, P.B. Farmer, F.J. van ¨ Dam, H. van Mossel, H.M. Schoemaker, S. Osterman-Golkar, Ch. Uebel, Y.S. Tang, A.H. Zwinderman, A.T. Natarajan, L. Ehrenberg, Biological and chemical monitoring of occupational exposure to ethylene oxide, Mutation Res. 250 Ž1991. 483–497. w11x P.A. Schulte, M. Boeniger, J.T. Walker, S.E. Schober, M.A. Pereira, D.K. Gulati, J.P. Wojciechowski, A. Garza, R. Froelich, G. Strauss, W.E. Halperin, R. Herrick, J. Griffith, Biologic markers in hospital workers exposed to low levels of ethylene oxide, Mutation Res. 278 Ž1992. 237–251. w12x D. Lerda, R. Rizzi, Cytogenetic study of persons occupationally exposed to ethylene oxide, Mutation Res. 281 Ž1992. 31–37. w13x E.C. Miller, J.A. Miller, Mechanisms of chemical carcinogenesis: nature of proximate carcinogens and interactions with macromolecules, Pharmacol. Rev. 18 Ž1966. 805–838. w14x V.E. Walker, T.R. Skopek, A mouse model for the study of in vivo mutational spectra: Sequence specificity of ethylene oxide at the hprt locus, Mutation Res. 288 Ž1993. 151–162. w15x D. Segerback, ¨ Reaction products in hemoglobin and DNA after in vitro treatment with ethylene oxide and N-Ž2-hydroxyethyl.-N-nitrosourea, Carcinogenesis 11 Ž1990. 307– 312. w16x F. Li, A. Segal, J.J. Solomon, In vitro reaction of ethylene oxide with DNA and characterization of DNA adducts, Chem.–Biol. Interact. 83 Ž1992. 35–54. w17x S. Osterman-Golkar, P.B. Farmer, D. Segerback, ¨ E. Bailey, C.J. Calleman, K. Svensson, L. Ehrenberg, Dosimetry of ethylene oxide in the rat by quantitation of alkylated histidine in hemoglobin, Teratogen. Carcinogen. Mutagen. 3 Ž1983. 395–405. w18x D. Segerback, ¨ Alkylation of DNA and haemoglobin in the mouse following exposure to ethane and ethene oxide, Chem.–Biol. Interact. 45 Ž1993. 139–151. w19x V.E. Walker, T.R. Fennell, P.B. Upton, T.R. Skopek, V. Prevost, D.E.G. Shuker, J.A. Swenberg, Molecular dosimetry of ethylene oxide: formation and persistence of 7-Ž2hydroxyethyl.guanine in DNA following repeated exposures of rats and mice, Cancer Res. 52 Ž1992. 4328–4334. w20x C.D. Brown, J.P. MacNeela, B. Wong, T.R. Fennell, DNA and hemoglobin adduct dosimetry in normal and transgenic mice exposed to ethylene oxide, Proc. Am. Assoc. Cancer Res. 35 Ž1994. 95. w21x V.E. Walker, T.R. Fennell, P.B. Upton, J.P. MacNeela, J.A. Swenberg, Molecular dosimetry of DNA and hemoglobin adducts in mice and rats exposed to ethylene oxide, Environ. Health Persp. 99 Ž1993. 11–17. w22x A. Kolman, T. Bohusova, ˇ ´ B. Lambert, J.W.I.M. Simons, Induction of 6-thioguanine-resistant mutants in human diploid fibroblasts in vitro with ethylene oxide, Environ. Mol. Mutagen. 19 Ž1992. 93–97. w23x T. Bastlova, ´ B. Andersson, B. Lambert, A. Kolman, Molecular analysis of ethylene oxide-induced mutations at the HPRT locus in human diploid fibroblasts, Mutation Res. 287 Ž1993. 283–292.

221

w24x J.E. Cochrane, T.R. Skopek, Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice, Carcinogenesis 15 Ž1994. 719–723. w25x Walker, V.E., N.J. Gorelick, J.A. Andrews, T.R Craft, J. deBoer, B.W. Glickman and T.R. Skopek Ž1996. Frequency and spectrum of ethylnitrosourea induced mutation at the hprt and lacI loci in splenic T-cells of exposed lacI transgenic mice, Cancer Res., in press. w26x Meng, Q., L. Recio, B.A. Wong, A. Reilly and V.E. Walker Ž1996. Comparison of the mutagenic potency of 1,3-butadiene at the hprt locus of T-cells following inhalation exposures of B6C3F1 mice and F344 rats, Carcinogenesis, submitted. w27x V.E. Walker, I.M. Jones, J. Nakamura, K. Burkhart-Schultz, T.R. Skopek, Age-dependent relationships between exposure and phenotypic expression of hprt mutations in T-cells from mice treated with N-ethyl-N-nitrosourea, Environ. Mol. Mutagen. 21 ŽSuppl. 22. Ž1993. 75. w28x I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea, Environ. Mutagen. 9 Ž1987. 317–329. w29x B.A. Smith, N.F. Fullerton, R.H. Heflich, F.A. Beland, DNA adduct formation and T-lymphocyte mutation induction in F344 rats implanted with tumorigenic doses of 1,6-dinitropyrene, Cancer Res. 55 Ž1995. 2316–2324. w30x Swenberg, J.A., Fedtke, N., Fennell, T.R. and Walker, V.E. Ž1990. Relationships between carcinogen exposure, DNA adducts and carcinogenesis, in: D.B. Clayson, I.C. Munro, P. Shubik and J.A. Swenberg ŽEds.., Progress in Predictive Toxicology, Elsevier, Amsterdam, pp. 161–184. w31x V.E. Walker, T.R. Craft, T.R. Skopek, Spectrum of ethylene oxide induced mutation in hprt exon 3 of splenic T-cells from exposed B6C3F1 mice, Environ. Mol. Mutagen. 23 ŽSuppl. 23. Ž1994. 72. w32x K.S. Tao, C. Urlando, J.A. Heddle, Comparison of somatic mutation in a transgenic versus host locus, Proc. Natl. Acad. Sci. USA 90 Ž1993. 10681–10685. w33x R.A. Winegar, L.H. Lutze, J.D. Hamer, K.G. O’Loughlin, J.C. Miralis, Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice, Mutation Res. 307 Ž1994. 479–487. w34x N.J. Gorelick, Overview of mutation assays in transgenic mice for routine testing, Environ. Mol. Mutagen. 25 Ž1995. 218–230. w35x A. Kolman, M. Naslund, C.J. Calleman, Genotoxic effects of ¨ ethylene oxide and their relevance to human cancer, Carcinogenesis 7 Ž1986. 1245–1250. w36x E. Agurell, H. Cederberg, L. Ehrenberg, K. Lindahl-Kiessling, U. Rannug, M. Tornqvist, Genotoxic effects of ethy¨ lene oxide and propylene oxide: a comparative study, Mutation Res. 250 Ž1991. 229–237. w37x T.R. Skopek, Of mice and men: target size and sensitivity, Mutation Res. 331 Ž1995. 225–228. w38x T.R. Skopek, K.L. Kort, D.R. Marino, Relative sensitivity of

222

V.E. Walker et al.r Mutation Research 392 (1997) 211–222

the endogenous hprt gene and lacI transgene in ENU-treated Big Blue e B6C3F1 mice, Environ. Mol. Mutagen. 26 Ž1995. 9–15. w39x Skopek, T.R., K.K. Kort, D.R. Marino, L.V. Mittal, D.R. Umbenhauer, G.M. Laws and S.P. Adams Ž1996. Mutagenic response of the endogenous hprt gene and lacI transgene in

benzow axpyrene-treated Big Blue w mice, Environ. Mol. Mutagen., submitted. w40x Walker, V.E., J.L. Andrews, P.B. Upton, J.G. deBoer and N.J. Gorelick Ž1996. Detection of cyclophosphamide-induced mutations at the hprt but not the lacI locus in splenic lymphocytes of exposed mice, Cancer Res., submitted.