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Mechanisms in cyclophosphamide induction of cytogenetic damage in human lymphocyte cultures
Mechanisms in Cyclophosphamide Induction of Cytogenetic Damage in Human Lymphocyte Cultures Linda M. Sargent, Boyd Roloff, and Lorraine F. Meisner
ABSTRACT: Low-dose cyclophosphamide treatment of human lymphocyte cultures in concentrations ranging from 0.001 to 0.00001 ~g/ml produced a statistically significant dose response in chromosome breakage and cell death. However, a dose as high as 0.2 p~g/ml did not produce significant damage in comparably treated whole blood cultures, These results suggest that
lymphocytes in culture have the ability to metabolize the nonmutagenic cyclophosphamide parent compound to its more mutagenic metabolite, but that such conversion may be prevented by binding of cyclophosphamide to red blood cells.
INTRODUCTION
Cyclophosphamide is an antitumor product that is not mutagenic unless metabolically activated to 4-hydroxycyclophosphamide [1-3]. The capability of cultured lymphocytes to activate cyclophosphamide has been suggested by studies showing an increase in sister chromatid exchange (SCE) induction in rat, rabbit, and human lymphocytes exposed in vitro [4,5], as well as decreased 3H thymidine incorporation and a reduction in mitotic index [6]. However, one recent study failed to detect chromosome damage following cyclophosphamide treatment of whole blood cultures from normal individuals [7]. The present study was undertaken in order to examine mechanisms in production of cytogenetic damage following in vitro exposure to cyclophosphamide. METHODS
Five-milliliter, 72-hour cultures using the blood from four to five donors were grown in 5 ml RPMI 1640 medium with 10% newborn calf serum, 100 U/ml penicillin, 100 ~g/ml streptomycin and 4 ~g/ml phytohemagglutinin (PHA). These cultures were treated with freshly diluted 99% pure cyclophosphamide (Sigma, St. Louis, MO) during the last 48 hours of culture, and harvested using standard techniques. Lymphocyte cultures were prepared with 1 ml of leukocyte-rich plasma in 4 ml of media, with 0.001-0.00001 ~g/ml cyclophosphamide added after 24 hours in culture. To evaluate the effect of red blood cells (RBC), cultures were also set up using 0.5 ml whole blood in 4.5 ml media, with 0.2 ~g/ml cyclophosphamide added From the University of Wisconsin, State Laboratory of Hygiene, Cytogenetics Section, Madison, Wl.
Address requests for reprints to Dr. Lorraine F. Meisner, University of Wisconsin, State Laboratory of Hygiene, Cytogenetics Sect., 465 Henry Mall, Madison, WI 53706. Received January 12, 1987; accepted April 20, 1987.
239 © 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genet Cytogenet 29:239-243(1987) 0165-4608/87/$03.50
240
L . M . S a r g e n t , B. Roloff, a n d L. F. M e i s n e r
after 24 h o u r s . I n a d d i t i o n , m i x e d c u l t u r e s w e r e p r e p a r e d u s i n g 0.5 m l of l e u k o c y t e r i c h p l a s m a p l u s 0.5 m l of g r a v i t y s e t t l e d RBC p e r 4 m l of m e d i a , a n d f o l l o w e d b y t r e a t m e n t w i t h 0.2 p,g/ml c y c l o p h o s p h a m i d e . The slides were coded, and then randomly scored by two different investigators. F r o m 200 to 400 cells w e r e e x a m i n e d f r o m e a c h c u l t u r e g r o w n at t h e s a m e t i m e , w i t h b l o o d f r o m at l e a s t f o u r d i f f e r e n t d o n o r s u s e d for e a c h d o s e p o i n t . E a c h cell w a s s c o r e d for c h r o m o s o m e b r e a k a g e as w e l l as for c h r o m o s o m e t y p e a b e r r a t i o n s c o n s i s t i n g of d i c e n t r i c c h r o m o s o m e s , t r a n s l o c a t i o n s , i n v e r s i o n s , a n d rings. M i t o t i c i n d e x w a s b a s e d o n t h e n u m b e r of d i v i d i n g cells p e r 1000 cells. I n d u c e d c h r o m o some breakage was determined by substracting the breakage observed in an individu a l ' s u n t r e a t e d c u l t u r e f r o m t h a t o b s e r v e d i n t h e i r t r e a t e d c u l t u r e s g r o w n at t h e same time.
RESULTS A d o s e - r e l a t e d i n c r e a s e i n c h r o m o s o m e b r e a k s ( r 2 = 0.986; y - 6.85 log X + 75.5) a n d a d e c r e a s e i n m i t o t i c i n d e x ( r 2 : 0.99; y = - 1 1 . 5 7 log X - 42.99) w a s obs e r v e d i n l y m p h o c y t e c u l t u r e s t r e a t e d w i t h 0 . 0 0 1 - 0 . 0 0 0 0 1 p,g/ml c y c l o p h o s p h a m i d e (p < 0.01) as s h o w n i n T a b l e 1 a n d F i g u r e 1. A t d o s e s of 0 . 0 1 - 0 . 2 ~ g / m l c y c l o p h o s p h a m i d e , t h e m i t o t i c i n d e x of t h e l y m p h o c y t e c u l t u r e s w a s too l o w to p e r m i t r e l i a b l e s c o r i n g . By c o n t r a s t , t h e w h o l e b l o o d c u l t u r e s t r e a t e d w i t h 0.2 ~g/ ml cyclophosphamide did not demonstrate statistically significant chromosome d a m a g e , w h e r e a s , t h e c u l t u r e s g r o w n w i t h 0.5 m l p l a s m a p l u s 0.5 m l w h o l e b l o o d h a d a n i n t e r m e d i a t e l e v e l of c h r o m o s o m e d a m a g e .
DISCUSSION Our results with the lymphocyte cultures indicate that there has been metabolic a c t i v a t i o n of c y c l o p h o s p h a m i d e b y l y m p h o c y t e s , b e c a u s e c y c l o p h o s p h a m i d e is k n o w n to b e i n a c t i v e u n l e s s m e t a b o l i z e d to its m u t a g e n i c m e t a b o l i t e s [1-3]. By contrast, the damage in the whole blood cultures was not statistically different from
Table 1
I n d u c t i o n of c y t o g e n e t i c d a m a g e i n c y c l o p h o s p h a m i d e - t r e a t e d c u l t u r e s
Cyclophosphamide concentration Lymphocyte cultures 0.001 p.g/m| 0.0005 p.g/ml 0.0001 p.g/ml 0.00001 ~g/ml Control Whole blood culture 0.2 p~g/ml Control Mixed cultures b 0.2 ~g/ml Control
Mean chromatid breakage (%)
Induced a chromatid breakage (%}
Chromosomal aberrations (%)
Mitotic Total cells index (%) scored
19.0 18.0 11.3 6.4 5.3
14.3 8.0 6.0 0.6 --
2.8 2.0 1.0 0.4 --
2.4 2.5 2.8 3.5 4.1
400 400 400 400 400
9.8 5.1
3.1 --
--
4.6 7.0
200 200
15.0 4.9
10.5 --
1.0 --
5.4 9.1
200 200
°Induced breakage reflects an average obtained by subtracting breakage observed in each individual's untreated culture from all treated cultures grown at the same time from the same individual. Results from four to five individuals make up this average. bMixed cultures used 0.5 ml plasma + 0.5 ml red cells per 5 ml medium, versus 0.5 ml of whole blood in whole blood cultures or 1 ml leukocyte-rich plasma in lymphocyte cultures.
241
Induction of Cytogenetic Damage
M°I. x
Breaks 0 20.
I00
18-
90
16-
80
0
14-
X
70
12-
60
i0-
50
8-
40
6-
30
4-
20
2-
i0
0
0
-11
-i0 Log cyclophosphamide g/ml
-9
Figure 1 Regression showing relationship of cytogenetic damage and mitotic depression to cyclophosphamide concentration in lymphocyte cultures. Breaks (O) refer to percent induced chromatid breakage in treated cultures; mitotic index (X) is expressed as percent of the related control mitotic index. untreated control cultures, suggesting that cyclophosphamide binding to RBC prevented metabolic activation. The lower chromosome damage in the mixed cultures, which had 50% RBC, further corroborates the role of RBC in the binding and inactivation of cyclophosphamide. The fact that the damage in the mixed culture was greater than the whole blood culture reflects the lower ratio of red blood cells to white blood cells (50:50) in the mixed cultures, compared with whole blood cultures in which we found an average of 70% of the volume (by gravity sedimentation) to be due to red cells. The observed cytogenetic damage associated with cyclophosphamide in lymphocyte cultures perhaps is due to the P450 system, which has been detected in PHAstimulated lymphocytes [8] and is capable of metabolizing cyclophosphamide [3,8]. Lymphocytes also have prostaglandin endoperoxide synthetase (PES) [11], an enzyme that has been proposed as an alternate pathway in the activation of cyclophosphamide by hepatoma cell lines that have no detectable P450 [9]. The findings in this study explain the unexpected results of a recent report by Joenje and Oostra, which reported elevated chromosomal breakage in whole blood cultures from patients with Fanconi's anemia but not in comparable cultures from normal controls [7]. However, as our results demonstrate, failure to observe chromosome damage in cyclophosphamide-treated whole blood cultures from normal volunteers could be explained by diminished metabolic activation as a result of cyclophosphamide complexing with erythrocytes present in the whole blood cultures. Perhaps the lower number of erythrocytes in the cultures from Fanconi's ane-
242
L.M. Sargent, B. Roloff, and L. F. Meisner
mia patients allowed more cyclophosphamide to be available for metabolic activation, accounting for the increased chromosomal damage reported in the study by Joenje and Oostra [7]. Similar erythrocyte b i n d i n g of lipophilic c o m p o u n d s and their metabolites was observed by Jontunen et al. [10] who saw much less damage in whole blood cultures exposed to vinylacetate compared with the increased damage seen in similarly treated lymphocyte cultures. However, Ray and Altenberg [12] found that the addition of red blood cells or red cell lysate increased the chromosome damage caused by selenite. The activation of selenite required oxygen and hemoglobin. The increased hydrogen peroxide, increased oxygen consumption, and reduced glutathione levels that occur during metabolism of selenite in the presence of red blood cells [13] are further evidence of radical generation. Metals such as selenium can be oxidized and reduced, and in the presence of oxygen can generate oxygen radicals. When both hydrogen peroxide and iron or free selenium are present (i.e., as in RBC or RBC lysate), the Haber Weis reaction would produce the even more reactive hydroxyl radical [14]. Because cyclophosphamide is not activated by a free radical mechanism, one would not expect the damage to be increased by RBC lysate or by the presence of RBC. The present study shows that adding cyclophosphamide to cultures of the leukocyte rich buffy layer containing few red cells results in dose-related production of chromosome damage, proving that lymphocytes, indeed, are capable of activation of cyclophosphamide in vitro without exogenous activation. The reasons for use of such low cyclophosphamide doses in this experiment was to prevent inactivation of the P450 enzymes, which has been shown to occur at high doses [6]. The fact that RBC evidently are able to complex with the cyclophosphamide emphasizes that such culture factors must be controlled if one wishes to draw any conclusions relative to the significance or a m o u n t of cyclophosphamide-induced chromosome breakage.
REFERENCES
1. Brock N (1976): Comparative pharmacologic study in vitro and in vivo with cyclophosphamide (NSC-26271), cyclophosphamide metabolites and plain nitrogen mustard compounds. Cancer Treat Rep 60:301-308. 2. Domeyer BE, Slade NE (1980): Kinetics of cyclophosphamide biotransformation in vivo. Cancer Res 40:174-180. 3. Hales BF, Jain J (1980): Characteristics of the activation of cyclophosphamide to a mutagen by rat liver. Biochem Pharmacol 29:256-259. 4. Waalkens DH (1981): Sister-chromatid exchanges induced in vitro by cyclophosphamide without exogenous metabolic activation in lymphocytes from three mamalian species. Toxicol Lett (Amst.) 7:229-232. 5. Wilmer JL, Erexon GL, Klingerman AD (1984): Sister chromatid exchange induction in mouse B and T lymphocytes exposed to cyclophosphamide in vitro and in vivol Cancer Res 44:880-884. 6. Sharma BS (1983): Effects of cyclophosphamide on in vitro human lymphocyte culture and mitogenic simulation. Transplantation 35:165-168. 7. Joenje H, Oostra AB (1986): Clastogenicity of cyclophosphamide in Fanconi's anemia lymphocytes without exogenous metabolic activation. Cancer Genet Cytogenet 22:339-345. 8. Song BV, Gelboin AV, Park SS (1984): Monoclonal antibody directed radioimmunoassay detects cytochrome P450 in human placenta and lymphocytes. Science 228:490-492. 9. Dearfield KL (1983): Evaluation of a human hepatoma cell line as a target cell in genetic toxicology. Mutat Res 108:437-439. 10. Jontunen K, Maki-Paakkanen J, Norppa H (1986): Induction of chromosome aberrations by stryene and vinylacetate in cultured human lymphocytes; dependence on erythrocytes. Mutat Res 159:109-116.
I n d u c t i o n of C y t o g e n e t i c D a m a g e
243
11. Homa ST, Conroy DM, Smith AD (1984): Unsaturated fatty acids stimulate the formation of lipoxygenase and cyclooxygenase products in rat spleen lymphocytes. Prost Leuk Med 14:417-27. 12. Ray JH, Altenburg L (1978): Sister chromatid exchange induction by sodium selenite: dependence on the presence of red blood cells or red blood cell lysate. Mutat Res 54:343354. 13. Sandholm, M (1973): The metabolism of selenite in cow blood in vitro. Acta Pharmacol Toxicol 33:6-16. 14. Bus JS, and Gibson JE (1984): Role of activated oxygen in chemical toxicity. In: Drug Metabolism and Drug Toxicology, Mitchell RJ, Horning GM, eds. Raven Press, NY, pp. 21-27.
ABSTRACT: Low-dose cyclophosphamide treatment of human lymphocyte cultures in concentrations ranging from 0.001 to 0.00001 ~g/ml produced a statistically significant dose response in chromosome breakage and cell death. However, a dose as high as 0.2 p~g/ml did not produce significant damage in comparably treated whole blood cultures, These results suggest that
lymphocytes in culture have the ability to metabolize the nonmutagenic cyclophosphamide parent compound to its more mutagenic metabolite, but that such conversion may be prevented by binding of cyclophosphamide to red blood cells.
INTRODUCTION
Cyclophosphamide is an antitumor product that is not mutagenic unless metabolically activated to 4-hydroxycyclophosphamide [1-3]. The capability of cultured lymphocytes to activate cyclophosphamide has been suggested by studies showing an increase in sister chromatid exchange (SCE) induction in rat, rabbit, and human lymphocytes exposed in vitro [4,5], as well as decreased 3H thymidine incorporation and a reduction in mitotic index [6]. However, one recent study failed to detect chromosome damage following cyclophosphamide treatment of whole blood cultures from normal individuals [7]. The present study was undertaken in order to examine mechanisms in production of cytogenetic damage following in vitro exposure to cyclophosphamide. METHODS
Five-milliliter, 72-hour cultures using the blood from four to five donors were grown in 5 ml RPMI 1640 medium with 10% newborn calf serum, 100 U/ml penicillin, 100 ~g/ml streptomycin and 4 ~g/ml phytohemagglutinin (PHA). These cultures were treated with freshly diluted 99% pure cyclophosphamide (Sigma, St. Louis, MO) during the last 48 hours of culture, and harvested using standard techniques. Lymphocyte cultures were prepared with 1 ml of leukocyte-rich plasma in 4 ml of media, with 0.001-0.00001 ~g/ml cyclophosphamide added after 24 hours in culture. To evaluate the effect of red blood cells (RBC), cultures were also set up using 0.5 ml whole blood in 4.5 ml media, with 0.2 ~g/ml cyclophosphamide added From the University of Wisconsin, State Laboratory of Hygiene, Cytogenetics Section, Madison, Wl.
Address requests for reprints to Dr. Lorraine F. Meisner, University of Wisconsin, State Laboratory of Hygiene, Cytogenetics Sect., 465 Henry Mall, Madison, WI 53706. Received January 12, 1987; accepted April 20, 1987.
239 © 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genet Cytogenet 29:239-243(1987) 0165-4608/87/$03.50
240
L . M . S a r g e n t , B. Roloff, a n d L. F. M e i s n e r
after 24 h o u r s . I n a d d i t i o n , m i x e d c u l t u r e s w e r e p r e p a r e d u s i n g 0.5 m l of l e u k o c y t e r i c h p l a s m a p l u s 0.5 m l of g r a v i t y s e t t l e d RBC p e r 4 m l of m e d i a , a n d f o l l o w e d b y t r e a t m e n t w i t h 0.2 p,g/ml c y c l o p h o s p h a m i d e . The slides were coded, and then randomly scored by two different investigators. F r o m 200 to 400 cells w e r e e x a m i n e d f r o m e a c h c u l t u r e g r o w n at t h e s a m e t i m e , w i t h b l o o d f r o m at l e a s t f o u r d i f f e r e n t d o n o r s u s e d for e a c h d o s e p o i n t . E a c h cell w a s s c o r e d for c h r o m o s o m e b r e a k a g e as w e l l as for c h r o m o s o m e t y p e a b e r r a t i o n s c o n s i s t i n g of d i c e n t r i c c h r o m o s o m e s , t r a n s l o c a t i o n s , i n v e r s i o n s , a n d rings. M i t o t i c i n d e x w a s b a s e d o n t h e n u m b e r of d i v i d i n g cells p e r 1000 cells. I n d u c e d c h r o m o some breakage was determined by substracting the breakage observed in an individu a l ' s u n t r e a t e d c u l t u r e f r o m t h a t o b s e r v e d i n t h e i r t r e a t e d c u l t u r e s g r o w n at t h e same time.
RESULTS A d o s e - r e l a t e d i n c r e a s e i n c h r o m o s o m e b r e a k s ( r 2 = 0.986; y - 6.85 log X + 75.5) a n d a d e c r e a s e i n m i t o t i c i n d e x ( r 2 : 0.99; y = - 1 1 . 5 7 log X - 42.99) w a s obs e r v e d i n l y m p h o c y t e c u l t u r e s t r e a t e d w i t h 0 . 0 0 1 - 0 . 0 0 0 0 1 p,g/ml c y c l o p h o s p h a m i d e (p < 0.01) as s h o w n i n T a b l e 1 a n d F i g u r e 1. A t d o s e s of 0 . 0 1 - 0 . 2 ~ g / m l c y c l o p h o s p h a m i d e , t h e m i t o t i c i n d e x of t h e l y m p h o c y t e c u l t u r e s w a s too l o w to p e r m i t r e l i a b l e s c o r i n g . By c o n t r a s t , t h e w h o l e b l o o d c u l t u r e s t r e a t e d w i t h 0.2 ~g/ ml cyclophosphamide did not demonstrate statistically significant chromosome d a m a g e , w h e r e a s , t h e c u l t u r e s g r o w n w i t h 0.5 m l p l a s m a p l u s 0.5 m l w h o l e b l o o d h a d a n i n t e r m e d i a t e l e v e l of c h r o m o s o m e d a m a g e .
DISCUSSION Our results with the lymphocyte cultures indicate that there has been metabolic a c t i v a t i o n of c y c l o p h o s p h a m i d e b y l y m p h o c y t e s , b e c a u s e c y c l o p h o s p h a m i d e is k n o w n to b e i n a c t i v e u n l e s s m e t a b o l i z e d to its m u t a g e n i c m e t a b o l i t e s [1-3]. By contrast, the damage in the whole blood cultures was not statistically different from
Table 1
I n d u c t i o n of c y t o g e n e t i c d a m a g e i n c y c l o p h o s p h a m i d e - t r e a t e d c u l t u r e s
Cyclophosphamide concentration Lymphocyte cultures 0.001 p.g/m| 0.0005 p.g/ml 0.0001 p.g/ml 0.00001 ~g/ml Control Whole blood culture 0.2 p~g/ml Control Mixed cultures b 0.2 ~g/ml Control
Mean chromatid breakage (%)
Induced a chromatid breakage (%}
Chromosomal aberrations (%)
Mitotic Total cells index (%) scored
19.0 18.0 11.3 6.4 5.3
14.3 8.0 6.0 0.6 --
2.8 2.0 1.0 0.4 --
2.4 2.5 2.8 3.5 4.1
400 400 400 400 400
9.8 5.1
3.1 --
--
4.6 7.0
200 200
15.0 4.9
10.5 --
1.0 --
5.4 9.1
200 200
°Induced breakage reflects an average obtained by subtracting breakage observed in each individual's untreated culture from all treated cultures grown at the same time from the same individual. Results from four to five individuals make up this average. bMixed cultures used 0.5 ml plasma + 0.5 ml red cells per 5 ml medium, versus 0.5 ml of whole blood in whole blood cultures or 1 ml leukocyte-rich plasma in lymphocyte cultures.
241
Induction of Cytogenetic Damage
M°I. x
Breaks 0 20.
I00
18-
90
16-
80
0
14-
X
70
12-
60
i0-
50
8-
40
6-
30
4-
20
2-
i0
0
0
-11
-i0 Log cyclophosphamide g/ml
-9
Figure 1 Regression showing relationship of cytogenetic damage and mitotic depression to cyclophosphamide concentration in lymphocyte cultures. Breaks (O) refer to percent induced chromatid breakage in treated cultures; mitotic index (X) is expressed as percent of the related control mitotic index. untreated control cultures, suggesting that cyclophosphamide binding to RBC prevented metabolic activation. The lower chromosome damage in the mixed cultures, which had 50% RBC, further corroborates the role of RBC in the binding and inactivation of cyclophosphamide. The fact that the damage in the mixed culture was greater than the whole blood culture reflects the lower ratio of red blood cells to white blood cells (50:50) in the mixed cultures, compared with whole blood cultures in which we found an average of 70% of the volume (by gravity sedimentation) to be due to red cells. The observed cytogenetic damage associated with cyclophosphamide in lymphocyte cultures perhaps is due to the P450 system, which has been detected in PHAstimulated lymphocytes [8] and is capable of metabolizing cyclophosphamide [3,8]. Lymphocytes also have prostaglandin endoperoxide synthetase (PES) [11], an enzyme that has been proposed as an alternate pathway in the activation of cyclophosphamide by hepatoma cell lines that have no detectable P450 [9]. The findings in this study explain the unexpected results of a recent report by Joenje and Oostra, which reported elevated chromosomal breakage in whole blood cultures from patients with Fanconi's anemia but not in comparable cultures from normal controls [7]. However, as our results demonstrate, failure to observe chromosome damage in cyclophosphamide-treated whole blood cultures from normal volunteers could be explained by diminished metabolic activation as a result of cyclophosphamide complexing with erythrocytes present in the whole blood cultures. Perhaps the lower number of erythrocytes in the cultures from Fanconi's ane-
242
L.M. Sargent, B. Roloff, and L. F. Meisner
mia patients allowed more cyclophosphamide to be available for metabolic activation, accounting for the increased chromosomal damage reported in the study by Joenje and Oostra [7]. Similar erythrocyte b i n d i n g of lipophilic c o m p o u n d s and their metabolites was observed by Jontunen et al. [10] who saw much less damage in whole blood cultures exposed to vinylacetate compared with the increased damage seen in similarly treated lymphocyte cultures. However, Ray and Altenberg [12] found that the addition of red blood cells or red cell lysate increased the chromosome damage caused by selenite. The activation of selenite required oxygen and hemoglobin. The increased hydrogen peroxide, increased oxygen consumption, and reduced glutathione levels that occur during metabolism of selenite in the presence of red blood cells [13] are further evidence of radical generation. Metals such as selenium can be oxidized and reduced, and in the presence of oxygen can generate oxygen radicals. When both hydrogen peroxide and iron or free selenium are present (i.e., as in RBC or RBC lysate), the Haber Weis reaction would produce the even more reactive hydroxyl radical [14]. Because cyclophosphamide is not activated by a free radical mechanism, one would not expect the damage to be increased by RBC lysate or by the presence of RBC. The present study shows that adding cyclophosphamide to cultures of the leukocyte rich buffy layer containing few red cells results in dose-related production of chromosome damage, proving that lymphocytes, indeed, are capable of activation of cyclophosphamide in vitro without exogenous activation. The reasons for use of such low cyclophosphamide doses in this experiment was to prevent inactivation of the P450 enzymes, which has been shown to occur at high doses [6]. The fact that RBC evidently are able to complex with the cyclophosphamide emphasizes that such culture factors must be controlled if one wishes to draw any conclusions relative to the significance or a m o u n t of cyclophosphamide-induced chromosome breakage.
REFERENCES
1. Brock N (1976): Comparative pharmacologic study in vitro and in vivo with cyclophosphamide (NSC-26271), cyclophosphamide metabolites and plain nitrogen mustard compounds. Cancer Treat Rep 60:301-308. 2. Domeyer BE, Slade NE (1980): Kinetics of cyclophosphamide biotransformation in vivo. Cancer Res 40:174-180. 3. Hales BF, Jain J (1980): Characteristics of the activation of cyclophosphamide to a mutagen by rat liver. Biochem Pharmacol 29:256-259. 4. Waalkens DH (1981): Sister-chromatid exchanges induced in vitro by cyclophosphamide without exogenous metabolic activation in lymphocytes from three mamalian species. Toxicol Lett (Amst.) 7:229-232. 5. Wilmer JL, Erexon GL, Klingerman AD (1984): Sister chromatid exchange induction in mouse B and T lymphocytes exposed to cyclophosphamide in vitro and in vivol Cancer Res 44:880-884. 6. Sharma BS (1983): Effects of cyclophosphamide on in vitro human lymphocyte culture and mitogenic simulation. Transplantation 35:165-168. 7. Joenje H, Oostra AB (1986): Clastogenicity of cyclophosphamide in Fanconi's anemia lymphocytes without exogenous metabolic activation. Cancer Genet Cytogenet 22:339-345. 8. Song BV, Gelboin AV, Park SS (1984): Monoclonal antibody directed radioimmunoassay detects cytochrome P450 in human placenta and lymphocytes. Science 228:490-492. 9. Dearfield KL (1983): Evaluation of a human hepatoma cell line as a target cell in genetic toxicology. Mutat Res 108:437-439. 10. Jontunen K, Maki-Paakkanen J, Norppa H (1986): Induction of chromosome aberrations by stryene and vinylacetate in cultured human lymphocytes; dependence on erythrocytes. Mutat Res 159:109-116.
I n d u c t i o n of C y t o g e n e t i c D a m a g e
243
11. Homa ST, Conroy DM, Smith AD (1984): Unsaturated fatty acids stimulate the formation of lipoxygenase and cyclooxygenase products in rat spleen lymphocytes. Prost Leuk Med 14:417-27. 12. Ray JH, Altenburg L (1978): Sister chromatid exchange induction by sodium selenite: dependence on the presence of red blood cells or red blood cell lysate. Mutat Res 54:343354. 13. Sandholm, M (1973): The metabolism of selenite in cow blood in vitro. Acta Pharmacol Toxicol 33:6-16. 14. Bus JS, and Gibson JE (1984): Role of activated oxygen in chemical toxicity. In: Drug Metabolism and Drug Toxicology, Mitchell RJ, Horning GM, eds. Raven Press, NY, pp. 21-27.