Sister Chromatid Exchange Induction in Mouse B- and T-Lymphocytes Exposed to Cyclophosphamide in Vitro and in Vivo1

[CANCER RESEARCH 44, 880-884, March 1984]

James L. Wilmer,2 Gregory L Erexson, and Andrew D. Kligerman

Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

ABSTRACT antibody responses in mice primed with sheep red blood cells, keyhole limpet hemocyanin, tetanus toxoid, and type III pneu- Cyclophosphamide (CPA) is known to exert greater toxic mococcal polysaccharide (23, 29, 31, 45, 47, 55); (d) greater, as effects on B- than on T-lymphocytes in vivo. Both in vitro and in well as more prolonged, impairment of B-cell response to mito- vivo CPA treatments were used to assess the possible cytoge- genie stimulation (47, 54); and (e) the destruction and longer netic basis for these observations. First, male C57BL/6 mouse regeneration time of a population of cells bearing surface immu- lymphocytes were stimulated to divide in vitro with either phy- noglobulin and exhibiting slow electrophoretic mobility (13). How tohemagglutinin (T-cell mitogen) or lipopolysaccharide (B-cell ever, other studies have focused on the disruption by CPA of mitogen), and were then treated with CPA (0.05 to 1.0 mw) and regulatory mechanisms involved in cell-mediated responses. For 5-bromo-2'-deoxyuridine (2 U.M)at 24 hr. Cultures were har example, the DTH response can be augmented by CPA (20 to vested at 60 hr following a 4-hr treatment with demecolcine (1.35 300 mg/kg) given before immunization (22, 23, 27, 32, 34, 42). MM).CPA caused concentration-related increases in sister chro- This augmentation was thought initially to be the result of release matid exchange (SCE) up to 3 times control frequencies; the of T-cells from Â¡mmunoregulatory factors secreted by B-suppres- resulting SCE induction curves for B- and T-cells were sigmoidal sor cells (22, 27); that is, a B-suppressor cell population was and equivalent. Second, mice were given a single i.p. injection being affected preferentially by CPA. Further studies demon of CPA (0.5, 1.0, or 5.0 mg/kg). Blood was removed 24 hr later strated that the augmented DTH response could occur even in and cultured without additional CPA, as described above. Dose- the presence of detectable antibody titers (4, 34). Evidence has related increases in SCE frequencies were seen for both T- and mounted that CPA is equally toxic to a T-suppressor cell popu B-lymphocytes. CPA induced consistently 2.5 to 3.7 more SCEs lation controlling the DTH response (23, 32, 38, 42). in B-cells than in T-cells. Thus, B- and T-lymphocytes exhibited Recently, a mouse lymphocyte culture assay has been devel an equal sensitivity to CPA in vitro, but B-cells were more oped in order to assess the cytogenetic effects of known muta- susceptible to the genotoxic effects in vivo. genie carcinogens and other environmental contaminants (14). Mouse peripheral blood lymphocytes can be stimulated to prolif INTRODUCTION erate with PHA (T-cell mitogen) (16, 46) and LPS (B-cell mitogen) (2,12,37). B-lymphocytes were found to have a characteristically CPA3 is known to exert toxic effects on mammalian hemato- lower SCE frequency, a longer cell cycle duration, and a higher poietic tissues. This toxicity is manifested as leukopenia (11,13, Ml than did T-lymphocytes. Because PHA-stimulated mammalian 29, 31, 50, 55), destruction of lymphocyte germinal centers in lymphocytes can metabolize CPA to reactive intermediates sub spleen and lymph nodes (47, 55), cytotoxic effects on hemic sequently leading to SCE induction (35, 52), it was possible that precursors (54), loss of humoral and cell-mediated immune func an innate difference between mouse B- and T-lymphocytes could tions (8, 29), cytogenetic damage (5, 24, 28), and cancer (3, 21, be demonstrated in vitro, based on the abilities of the cells to 41). Initial reports indicated that CPA administered in sublethal activate CPA. Furthermore, it was of interest to use the mouse doses (300 to 400 mg/kg) selectively depleted areas of mouse lymphocyte culture assay to determine whether there was a lymph nodes and spleen containing lymphocytes which are not possible cytogenetic basis for the observations of greater B-cell dependent on thymus integrity in late fetal or early neonatal life sensitivity to CPA in vivo. (47, 50). Thus, B-lymphocyte populations appeared to be dam aged preferentially compared to T-lymphocytes. Five general MATERIALS AND METHODS observations provided further support for the idea that short lived, rapidly dividing B-cells were more sensitive to CPA follow Animals. Male C57BL/6NCrlBr mice (6 to 8 weeks old) were obtained ing in vivo treatment: (a) a parallel increase in the relative pro from Charles River Breeding Laboratories (Kingston, NY) and were portion of lymphocytes bearing the thymus-derived 8 antigen placed in quarantine for 2 weeks prior to experimentation. The housing (39); (b) the direct toxic effect on lymphocytes during DMA conditions and diet were as described previously (14). synthesis (11, 27, 51 ); (c) severe depression of primary humoral CPA Exposure in Mitro. For each replicate experiment, 6 to 8 mice were exsanguinated by cardiac puncture while under methoxyflurane anesthesia. The blood was pooled and centrifuged in 6-ml Falcon culture 1This research was funded by the Chemical Industry Institute of Toxicology, a tubes for 7 min at 700 x g. The separated plasma was sterilized through privately funded institute. 2To whom requests for reprints should be addressed, at the Chemical Industry Millex GS filters (0.22 Mm; Millipore Corp., Bedford, MA). Complete medium was composed of RPMI medium 1640 plus 25 mw A/-2-hydrox- Institute of Toxicology, Department of Genetic Toxicology, P. O. Box 12137, yethylpiperazine-N'-2-ethanesulfonic acid buffer with L-glutamine Research Triangle Park, NC 27709. 'The abbreviations used are: CPA, Cyclophosphamide; SCE, sister chromatid (GIBCO), 20% heat-inactivated fetal calf serum (GIBCO), 6% pooled exchange; DTH, delayed-type hypersensitivity; Ml, mitotic index; PHA, phytohe- mouse plasma, 100 units penicillin, and 100 ^g streptomycin (GIBCO) magglutinin; LPS. lipopolysaccharide; GIBCO, Grand Island Biological Co.. Grand per ml, an additional 292 ^g L-glutamine (GIBCO) per ml, 10 units Island, NY. Received September 21, 1983; accepted November 11, 1983. preservative-free sodium heparin (The Upjohn Co., Kalamazoo, Ml) per

880 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1984 American Association for Cancer Research. CPA-induced SCE in Mouse Lymphocytes ml, and either 6 ng PHA (HA 16, Burroughs Wellcome and Co., Greenville, 20 NC) or 60 tig LPS (Escherichia coli serotype 0111 :B4; Sigma Chemical Co., St. Louis, MO) per ml. The blood cell pellet was washed 3 times with Dulbecco's phosphate-buffered saline (pH 7.5) supplemented with 18 2% heat-inactivated fetal calf serum, and resuspended to the initial whole blood volume in complete medium without mitogen. Total leukocyte 16 counts were determined from a 20-/il aliquot of the cell suspension on a Model 2, Coulter Counter (Coulter Electronics, Inc., Hialeah, FL). Aliquots containing 5.0 x 10s leukocytes were inoculated into culture tubes 14 containing approximately 0.9 ml complete medium. The aliquot volumes varied from 73 to 147 p\, depending on the replicate experiment. The cultures were incubated for 24 hr in a humidified 5% C02 atmosphere at 0. 37Â°.At 24 hr the cultures were centrifugea for 7 min at 700 x g. The original medium was replaced with 0.9 ml of fresh complete medium 10 (without mouse plasma) containing 2 ?M 5-bromo-2'-deoxyuridine (Sigma Chemical Co.) (14). CPA monohydrate (>98% pure; Aldrich Chemical Co., Inc., Milwaukee, Wl) was dissolved in RPMI Medium 1640 to a 100 U mW concentration and was filter sterilized. A constant 10-iil volume of CPA dilutions was added to the cultures to attain final concentrations of 0,0.05,0.1,0.25,0.5,0.75, and 1.0 mw. The culture tubes were wrapped in aluminum foil and returned to the incubator. After a further culture period of 32 hr, Colcemid (0.5 ^g/ml; GIBCO) was added to the cultures. B-CELLS Four hr later the cells were harvested and slides were prepared (26). T-CEUS The 60-hr harvest time allowed a greater proportion of cells to reach second division in the LPS-stimulated cultures, and took into considera tion potential cell cycle delay and mitotic lag induced by CPA. At least 4000 nuclei, 400 metaphases, and 70 diploid second-division meta- 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 09 1.0 phases per CPA concentration were analyzed from coded slides from 3 to 4 independent experiments for Ml, cell cycle kinetics, and SCE, CYCLOPHOSPHAMIDE (mM) respectively. Chart 1. SCE induction by CPA in vitro in mouse peripheral Wood lymphocytes CPA Exposure in Vivo. CPA was dissolved in RPMI Medium 1640 stimulated with LPS or PHA. Blood removal, lymphocyte culture, CPA treatment, and cell harvest were as described in "Materials and Methods." Points, means from and filter sterilized. Each mouse received a single i.p. injection of either 70 to 114 second-division metaphases from 3 (B-cell) or 4 (T-cell) independent 0.5, 1.0, or 5.0 mg CPA/kg or solvent in a carrier volume of about 0.3 experiments; oars, S.E. ml. The body weights varied from 26 to 34 g, but for each experiment the standard deviation was <10%. Because of the number of replicate concentration of CPA (0.05 ITIM)in T-lymphocytes, and no further cultures required, only one dose level of CPA could be used in any given depression in the Ml was observed with higher CPA concentra experiment. Therefore, 3 to 4 mice were given injections of CPA, and an tions. However, CPA concentrations >0.75 mM were needed to equal number of mice were used as concurrent controls. Blood was reduce the Ml in B-lymphocytes by 50%. With the exception of removed by cardiac puncture 24 hr after dosing and was processed separately for each donor. Whole blood cultures were established as one anomalous data point at 0.5 ITIMCPA, the B-cells exhibited described above for in vitro controls. Generally, 3 cultures per mitogen a more gradual, concentration-related decrease in Ml than did T- were initiated for each treated and control mouse. Cell harvest and cells. On the other hand, CPA had only minor effects on cell cytological preparations were as described above. Unless noted other cycle kinetics of B- and T-lymphocytes, as assessed by the wise, 1000 nuclei, 100 metaphases, and 25 second-division metaphases relative proportion of metaphases showing cell cycle-specific were scored per treatment for Ml, cell cycle progression, and SCE, staining patterns (Table 1). A uniformly gradual slowing of the respectively. cell cycle was discernible only with T-cells, where the proportion Statistical Treatment of the Data. The SCE frequency means from of fourth-division (and greater) metaphases dropped from 15.3% in vivo and in vitro CPA exposures were subjected to square root transformation to equalize variances (44). Comparisons between treated in control cultures to 7.1% in cultures exposed to 1 mM CPA. and control groups were done using Student's f test (one-tailed), and This delay in cell cycle time was statistically significant. the significance level of 0.05 was chosen. The Mis and cell cycle kinetics In vivo exposure to CPA resulted in significant, dose-related of lymphocytes were subjected to one-way analyses of variance. increases in the SCE frequencies of mouse peripheral blood T- and B-lymphocytes (Table 2). However, B-lymphocytes exhibited RESULTS consistently higher induced SCE frequencies in 3 separate ex periments. For example, the SCE frequency in B-cells from mice CPA induced concentration-related increases in the SCE fre dosed with CPA (5 mg/kg) was 3 times that of concurrent quency of up to 3 times control in LPS- and PHA-stimulated controls, whereas the relative increase in the SCE frequency in mouse lymphocytes during a 36-hr exposure in vitro (Chart 1). T-cells was only 2 times that of controls. Furthermore, the Small, but statistically significant elevations in SCE frequencies induced SCE frequency at 5 mg CPA/kg was 11.2 and 7.5 for were detected at 0.05 mw CPA in B- and T-lymphocytes. Al B- and T-lymphocytes, respectively. Thus, the difference be though control B- and T-cells had different base-line SCE fre tween the induced SCEs in B- and T-cells for doses of 0.5,1.0, quencies, the CPA-induced cytogenetic damage was essentially and 5.0 mg CPA/kg was 2.5, 3.3, and 3.7 SCEs/metaphase, equivalent for both cell populations over a 20-fold concentration respectively. Although a small, but statistically significant in range. Likewise, the Mis were depressed in cultures treated with crease in the SCE frequency of B-cells could be demonstrated CPA (Table 1). The Ml declined by about 50% at the lowest with the lowest CPA dose (0.5 mg/kg), the SCE frequency in

MARCH 1984 881

Table 1 Effect of CPA on the Ml and cell cycle kinetics of mouse peripheral blood B- and T-lymphocytes exposed in vitro. Blood removal, lymphocyte culture, CPA treatment, and cell harvest were as described in "Materials and Methods." At least 4000 nuclei were scored for Ml for each datum. Cell cycle kinetics were determined from at least 400 metaphases classified into cell cycle-specific staining patterns per mitogen.

Cell cycle kinetics (%)

MitogenPHA (mm)00.050.100.250.500.751.0000.050.100.250.500.751.00Ml(%)2.2 division27.8 division25.5 division31.4 division15.3 (T-cell)LPS(B-cetl)CPA *1 Â±0.6a- +2.426.2 +1.527.6 Â±1.835.0 Â±2.2e11.2 .2 Â±0.31.2 Â±2.130.8 Â±1.525.0 Â±1.833.1 Â±1.611.1 Â±0.31.0 Â±2.126.4 Â±1.329.1 +0.433.7 Â±1.910.8 Â±0.21.4 +2.030.7 +1.525.4+1.931.5 Â±1.433.3 Â±1.210.6 +0.31 Â±3.129.3 Â±2.729.5 Â±1.39.7 .2 Â±0.31.0 Â±2.831.1 +2.332.4 Â±1.729.4 Â±1.77.1 Â±0.25.6 Â±2.929.6 +3.140.0 Â±2.722.6 Â±1.67.8

Â±0.9"4.6 Â±1.227.8 Â±3.139.2 Â±2.423.8 Â±1.2"9.2 Â±0.44.3 Â±2.827.4 Â±4.443.6 Â±1.022.6 Â±1.36.4 +0.43.8 Â±1.425.8 Â±2.540.8 +2.026.5 Â±2.26.9 Â±0.45.2 Â±1.931.0 Â±1.140.8 +1.224.6 Â±1.03.6 Â±1.02.8 +1.829.8 Â±2.042.8 +1.519.8 +1.07.6 +0.63.2 Â±1.330.4 Â±2.146.0 +2.119.4 Â±1.74.2 Â±0.6First Â±2.1Second Â±4.2Third Â±3.1aFourth Â±2.2 " Mean Â±S.E. " Not significantly different at p < 0.05, using one-way analysisof variance. c Significantlydifferent at p < 0.05, using one-way analysisof variance.

Table 2 B- and T-lymphocytes were nearly identical and parallel, which SCE induction by CPA in vivo in mouse peripheral blood lymphocytes stimulated indicated that these 2 major cell populations were activating CPA with LPS and PHA. to about the same extent. However, analyses of cell cycle The injection of CPA, blood removal, lymphocyte culture, and cell harvest were as described in 'Materials and Methods.' progression showed that T-lymphocytes were slightly more sen

Difference sitive to CPA in vitro. Although no innate difference in SCE between induction to CPA could be demonstrated in B- and T-cells in Induced induced vitro, B-cells were consistently more susceptible to the effects CPA(mg/ Animals SCEs/meta- SCEs/ SCEs in B- Mitogen kg) (n) phase metaphase and T-cells of low doses of CPA in vivo, as shown by higher induced SCE LPS(B-ceH)PHA Â±0.1"8.3 frequencies (Table 2). Thus, these data demonstrate a cytoge- Â±0.1*7.8 netic basis for the observations that B-lymphocytes are more

(T-cell)LPS Â±0.27.8 sensitive to the effects of CPA in vivo. +0.36.2 Whole blood lymphocyte cultures from humans, rabbits, and (B-cell)PHA Â±0.1 rats have been shown to metabolize CPA to reactive intermedi 11.4 Â±0.2"8.3 1.001.005.005.04333333e433335.8 ates capable of inducing SCE (35, 52). Lymphocytes from these species were stimulated with PHA and exhibited elevations of (T-cell)LPS +0.110^0.2"5.1 SCE frequencies varying from about 1.6- to 6-fold above base line at 1 FTIMCPA, depending on the species and duration of (B-cell)PHA Â±0.016^0.0*7.3 cultures. CPA was present in the human lymphocyte cultures for 48 hr (35, 52), while rat and rabbit cultures were exposed for 76 (T-cell)00.500.50 +0.214.8 Â±0.4"2.50.05.21.911.27.52.53.33.7 hr (52). Another study has shown that CPA suppresses [3H] " Mean Â±S.E. thymidine incorporation in PHA-stimulated human lymphocytes 6 Significantly different from concurrent control at p < 0.05, using Student's f (43). This is also an indication of cytotoxic effects being mediated test (one-tailed). c Fifteen cells (instead of 25) analyzed from one mouse. by CPA metabolites. In contrast, no increase in the SCE fre quency was observed in human lymphocytes cultured for the CPA-exposed T-cells was the same as controls. CPA had no final 27 hr of a 75-hr culture in the presence of CPA concentra significant effects on cell cycle progression of B- and T-lympho tions as high as 1.8 HIM(1). Similarly, human lymphocytes treated cytes at any dose level (Table 3). Similarly, the Ml in T-cells was with 0.1 to 1.0 mM CPA for 1 to 2 hr at either 48 or 51 hr after not affected by any dose of CPA (Table 3). However, in B-cells mitogenic stimulation exhibited no increase in the SCE frequency the Ml was doubled at the 2 lowest doses of CPA, whereas no (7, 53). However, these short exposures were probably insuffi difference from control was observed at 5 mg CPA/kg. It should cient for any appreciable activation of CPA. Thus, there is be noted that no significant, dose-related decline in total leuko increasing evidence that mammalian lymphocytes can metabo cyte counts occurred following CPA treatment when peripheral lize carcinogens such as benzo(a)pyrene (40, 48), aflatoxin Bi (20, 49), 2-aminofluorene (49), urethan (7), and CPA (35, 43, 52) blood samples were taken from mice at 24 hr (data not shown). to genotoxic intermediates capable of inducing SCEs, although liver S-9 mix (30,49), microsomes (10), and hepatocyte-mediated DISCUSSION activation (25) are much more efficient. Furthermore, lympho These results demonstrate that mouse peripheral blood lym cytes appear to be as capable as hepatoma (9,19) or esophageal phocytes can metabolize CPA, as evidenced by the increase in (19) cell lines in activating CPA to SCE-inducing intermediates the SCE frequency and general suppression of cell proliferation over comparable exposure periods. Because fresh lymphocytes (Chart 1; Table 1). The SCE concentration-response curves for and some lymphoblastoid lines retain detectable levels of cyto-

882 CANCER RESEARCH VOL. 44

Tabte3 Effect of CPA on the Ml and cell cycle kinetics of mouse peripheral blood B- and T-lymphocytes exposed in vivo. The injection of CPA, Wood removal, lymphocyte culture, and cell harvest were as described in "Materials and Methods."

(%)MitogenLPS Cell cycle kinetics

(mg/kg)00.50 (n)4 (%)5.9 division29.0 division49.8 division19.8 division1.4 (B-cell)PHA Â±0.5a Â±4.2 Â±2.3 Â±2.7 Â±0.6 12.7 Â±0.5b2.8 33 25.0 Â±2.017.0 48.3Â±1.420.7 24.0Â±1.538.7 2.7Â±1.223.6

(T-cell)LPS Â±0.6 Â±2.0 Â±3.2 Â±2.3 Â±2.4 0.501.00 33 3.0 Â±0.24.7 17.3 Â±1.524.7 21.3 Â±0.943.7 33.7Â±1.926.0 27.7 Â±0.75.6

(B-cell)PHA Â±1.0 Â±5.8 Â±2.6 Â±5.6 Â±2.0 9.0 Â±0.2"2.7 334333 27.3 Â±2.916.7 48.0 +4.019.7 21.3 +2.431 3.4Â±1.331.9

(T-cell)LPS Â±0.4 Â±2.2 Â±0.9 .7 Â±4.3 Â±1.7 1.00 2.7 Â±0.66.7 13.8 +1.923.7 19.3+1.952.0 33.5 +2.122.0 33.4 Â±2.22.3

(B-cell)PHA Â±0.7 + 4.7 Â±2.0 Â±2.7 Â±1.2 5.00 5.6 +1.83.2 23.7 Â±5.212.3 47.0 Â±4.223.3 22.7 Â±2.729.7 6.6 Â±3.634.7

(T-cell)CPA + 0.3 Â±3.2 Â±2.7 Â±1.8 Â±2.0 5.0Animals 3Ml 2.7 Â±0.7First 12.3 Â±1.9Second 25.3 Â±0.7Third 31.0 Â±2.1aFourth 31.4 Â±1.3 a Mean + S.E. 6 Significantly different from concurrent control at p < 0.05, using Student's t test (one-tailed). chrome P-450 enzymes (17), and CPA is activated by a pheno- greater capabilities to metabolize CPA, or differential DNA repair barbital-induced form of hepatic cytochrome P-450 (18), it seems capabilities. Repair of DNA damage appears to be minimal in reasonable that these enzymes are responsible for biotransfor lymphocytes. For example, when lymphocytes were obtained mation of CPA in mitogen-stimulated B- and T-lymphocytes. from a cancer patient treated 24 hr earlier with CPA and placed Contributions from other enzyme systems, such as prostaglandin under liquid holding conditions for 12 to 72 hr prior to PHA endoperoxide synthetase, has been suggested recently as a stimulation, SCE induction was not diminished (36). This lack of possible alternate pathway for CPA metabolism in hepatoma cell reduction in SCE indicated that substantial DNA repair was not lines, where no cytochrome P-450 was detected (9). Nonspecific occurring either in the resting state (G0) or during blastogenesis. oxidation via active oxygen metabolites secreted by mitogen- The variable proliferative response of B-lymphocytes to LPS stimulated macrophages and polymorphonuclear leukocytes (15) stimulation after CPA exposure is paradoxical. For example, no could also account partly for CPA metabolism in whole-blood change in the Ml from control was observed at 5 mg CPA/kg cultures. The present data indicate that the metabolic capabilities (Table 3). However, the Ml was increased 2-fold at 0.5 and 1.0 of B- and T-lymphocytes in vitro are similar, as shown by the mg CPA/kg to over 9%. Because the historical and the present equivalent induction of SCE (Chart 1). control Mis are similar, the elevated Ml in the CPA treatment There is no doubt that cell-mediated and humoral immune groups is unusual. The mechanism of enhanced mitogenic re responses can be severely affected by CPA treatment in vivo (8, sponsiveness in B-cells after low-dose CPA treatment is not well 29), but the greater susceptibility of B-lymphocytes to SCE defined. One possible explanation is that CPA may be damaging induction parallels findings in other studies, where different end- a suppressor cell population which normally controls B-cell pro point responses were assessed. These altered responses in liferation during LPS exposure in vitro. This explanation is anal clude pronounced histopathological changes in B-cell popula ogous to the demonstration of a CPA-sensitive, T-suppressor tions in spleen and lymph nodes (47, 50), reduced ability to cell population which regulates cell-mediated immunity in the proliferate after mitogenic stimulation (13, 31, 47, 54), and dimi DTH response (23, 32, 42). The fact that the B-cell proliferative nution of primary humoral antibody response (23, 29, 31, 45, 47, response is enhanced has implications for the treatment of 55). Thus, these toxic effects occurring in concert suggest that tumors that are weakly immunogenic. Also, the lowered host SCE induction may be correlated with the loss of immune func resistance to pathogenic organisms and challenge with second tion in vivo. However, the cytogenetic results were obtained after ary tumors following high therapeutic doses of CPA might be low-dose CPA treatment (0.5 to 5.0 mg/kg), compatible with the obviated by the use of low doses, coupled with potentiated sensitivity of the SCE assay (i.e., no demonstrable cytotoxicity), humoral or cell-mediated immune reactions (6, 33). whereas most immunological studies used doses ranging from In conclusion, the mouse B- and T-lymphocyte culture tech 14 to 400 mg/kg. nique allows the examination of cytogenetic and cytotoxic effects The mechanism for the selective effect of CPA toxicity on B- of chemicals on 2 major cell populations responsible for immune cells is incompletely understood. Although it is known that CPA function. Thus, selective toxicity can be ascertained after in vivo metabolites exert direct toxic effects on dividing cells in vivo (11, or In vitro exposure. This technique is applicable to the study of 27, 51), the results presented here on selective SCE induction potential hematopoietic genotoxins and to the development of in normally nondividing peripheral blood lymphocytes suggest cell-specific chemotherapy. that other factors are operative in vivo. Some possible factors might include distribution of active metabolites to areas of lymph- ACKNOWLEDGMENTS oid tissues relatively enriched for B-cells, site-specific activation of CPA in rapidly proliferating B-cell germinal centers, association The authors thank Ed Bermudez, Craig Boreiko, and Michael Murray for critical review of the manuscript, and also Linda Smith and Joanne Quate for typing the of B-cells during peripheral blood circulation with cells possessing manuscript.

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884 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1984 American Association for Cancer Research. Sister Chromatid Exchange Induction in Mouse B- and T-Lymphocytes Exposed to Cyclophosphamide in Vitro and in Vivo

James L. Wilmer, Gregory L. Erexson and Andrew D. Kligerman

Cancer Res 1984;44:880-884.

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