93

Mutation Research, 248 (1991) 93-99

© 1991 ElsevierSciencePublishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100098K MUT 04962

Evidence for acrolein-modified D N A in peripheral blood leukocytes of cancer patients treated with cyclophosphamide Melissa A. McDiarmid a, p. Thomas Iype b, Ken Kolodner a, David Jacobson-Kram c a n d P a u l T. Strickland a "Johns Hopkins University, School of I-Iygiene and Public Health, Baltimore, MD 21205, b Biologic Research Faculty and Facility, Inc., Ijamsville, MD 21754 and c Microbiological Associates, Rockville, MD 20850 (U.S.A.)

(Received21 June 1990) (Revision received16 October 1990) (Accepted 18 October 1990)

Keywords: Acrolein; DNA, acrolein-modified;Leukocytes,peripheral blood; Cancer patients; Cyclophosphamide

Summary Monitoring human populations for specific D N A modifications has been made possible by developing highly sensitive immunoassays employing antibodies specific for carcinogen-DNA adducts. While these techniques have been used to follow occupationally and environmentally exposed populations, results have been limited by the lack of exposure data with which to correlate adduct formation. Cancer patients treated with precisely known doses of anticancer drugs can be studied to examine the association between drug dose and adduct formation. This study examined acrolein-modified D N A in patients treated with the anticancer drug cyclophosphamide (CP) and in newly diagnosed patients prior to treatment. Employing 2 different detection methods, enzyme-linked immunosorbent assay (ELISA) and immuno-dot blot (IDB), acrolein-modified D N A was identified in a total of 6 of 12 (50%) treated patients and in 0 of 15 untreated patients. Formation of acrolein-modified D N A was examined as a function of lifetime CP dose, recent CP dose, time since last treatment, regime of treatment, and smoking history; however no clear trends were observed.

Cyclophosphamide (CP), a commonly used chemotherapeutic alkylating drug is the principal agent for treatment of breast cancer (Carter, 1976), some hematopoietic and lymphatic cancers (Korst et al., 1964), and other malignancies (Benjamin et al., 1978). CP is a known human carcinogen asso-

Correspondence: Melissa A. McDiarmid, M.D., M.P.H., Johns Hopkins University,Schoolof Hygieneand Public Health, 615 N. Wolfe Street, Baltimore, MD 21205 (U.S.A.).

ciated with secondary malignancies, usually myelogenous leukemias developing in some patients previously treated with CP for a different primary cancer (Chabner, 1977; Hoover et al., 1981; Kaldor et al., 1990). The cytotoxic and carcinogenic properties of CP are believed to be due to its covalent interaction with cellular D N A (Vu et al., 1981). D N A adduct formation is considered to be an early, necessary event in carcinogenesis (Yuspa and Poirier, 1988; Beland and Poirier, 1989) but may not be sufficient to allow

94

CH 2 = CH - CHO AcroJeirl

CI.CH 2.CH 2

NH 2 X /

/

N-P=O \

CI.CH 2.CH 2

OH

Phosphoramide Mustard

CI.CH 2.CH 2

Methods

\ /

validation of the adduct measurement technique (Poirier et al., 1989). In this study, we examined acrolein-modified DNA formation in peripheral blood leukocytes (PBL) from cancer patients previously treated with CP and in newly diagnosed cancer patients prior to treatment.

NH

CI.CH 2.CH 2 Nomitrogen Mustard

Fig. 1. Cytotoxicproducts of cyclophosphamidemetabolism.

progression to malignancy without additional changes. CP must be metabolically activated in vivo with 2 principal metabolites resulting: phosphoramide mustard (PM) and acrolein (Fig. 1). PM is thought to be responsible for the drug's anti-cancer activity following dephosphoramidation to nornitrogen mustard (Fig. 1) (Colvin et al., 1976; Connors, 1978). Acrolein, a highly reactive aldehyde, may cause the CP-associated bladder cancer seen in some treated patients (Schmahl and Habs, 1983; Irving et al., 1987; IARC, 1987). Both PM (Benson et al., 1988) and acrolein (Foiles et al., 1989) form DNA adducts in vitro. The development of highly sensitive immunoassays employing antibodies specific for carcinog e n - D N A adducts has made possible the monitoring of DNA adducts in human populations (Poirier, 1984; Muller and Rajewsky, 1981; Strickland and Boyle, 1984). While this approach has been exploited in the evaluation of occupationally and environmentally exposed populations (Shamsuddin et al., 1985; Perera et al., 1988; Liou et al., 1989) these studies have been limited by the lack of exposure data with which to correlate adduct formation. A population whose exposure dose is known precisely would therefore be valuable in examining the association between exposure and adduct formation. Cancer patients treated with anticancer drugs provide such a population. Beyond the advantage of knowing exact exposure dose, which allows investigation of the dose-response relationship for adduct formation, the availability of unexposed controls allows further

Blood specimens were collected in vacutainer tubes containing lithium heparin from 27 cancer patients who had primarily breast and hematological malignancies. 12 patients had been therapeutically treated previously with a regime which included CP while 15 patients who were newly diagnosed and untreated at the time of analysis served as controls. All subjects gave informed consent for venipuncture and completed a detailed questionnaire covering past medical history, family history, smoking, alcohol, diet, medication and lifetime occupational history. All the treated patients had been on combination chemotherapy including CP or on CP alone. Breast cancer patients usually received intravenous (i.v.) CP therapy once a month. Other patients may have received monthly i.v. therapy or daily CP orally. 35 ml of blood was obtained by venipuncture and centrifuged (30 min, 2000 × g, 25°C); and nucleated PBL were collected and frozen at - 7 0 °C for DNA extraction or used immediately for sister-chromatid exchange analysis (McDiarmid et al., 1990). DNA was isolated from PBL samples by a modification of the method of Miller et al. (1988) involving salt fractionation followed by chloroform isoamyl alcohol extraction. Acrolein-modified DNA was detected by enzyme-linked immunosorbent assay (ELISA) and immunodot blot (IDB) assay using monoclonal antibody MoAb5 specific for acrolein-modified DNA (Shivapurkar et al., 1989). This antibody was raised against DNA modified with acrolein and conjugated with methylated bovine serum albumin. MoAb5 recognizes acrolein-modified DNA (either heat-denatured or native) with highest affinity. acrolein-modified deoxyguanosine with low affinity, but does not cross-react with unmodified DNA or nornitrogen mustard-modified DNA (Iype et al., in preparation).

95 washing the m e m b r a n e twice as above, it was incubated with alkaline phosphatase-conjugated goat anti-mouse I g G (Vectastain) for 1 h at RT. After washing the membrane twice with T w e e n / T B S and once with TBS, it was incubated with alkaline phosphatase substrate (Vectastain) in sodium bicarbonate buffer for 20 rain. Fisher's exact test was used to compare the occurrence of the detection of acrolein-modified D N A in the CP-treated group to the untreated control group. The Wilcoxon rank-sum test was used to compare those who did and did not develop acrolein-modified D N A among the CPtreated group for differences in CP dose, the time since CP dose and white blood cell counts. Due to the small sample size, Fisher's exact and the Wilcoxon rank-sum tests are preferred over the parametric alternatives (Snedecor and Cochran, 1976).

A direct-binding ELISA was used in which 10 (2-fold) serial dilutions of test D N A were coated on microtiter plates (2 h at 37 ° C) resulting in a range of 5 /xg-9.8 ng D N A / w e l l . Plates were blocked with 2 0 0 / d / w e l l of 1% gelatin in water (1 h at 37 ° C) and washed 3 times with 0.05% Tween in phosphate-buffered saline (PBS). Monoclonal antibody MoAb5 was diluted in 0.1% gelatin in PBS and added to wells at a final concentration of 20 ng antibody per well. After incubation (1 h at 37 ° C) the plates were washed 3 times as above. Biotinylated goat anti-mouse I g G (Vectastain) diluted in 0.1% gelatin in PBS was added to plates ( 5 0 / t g / w e l l ) and incubated as above. After washing plates 3 times as above, avidin-biotin conjugate-labeled alkaline phosphatase (Vectastain) diluted in 0.05% Tween PBS was added (50 ~tg/well) and incubated (0.5 h at 37°C). After washing plates 5 times as above, alkaline phosphatase substrate (Sigma) at a concentration of 1 m g / m l in sodium bicarbonate buffer (pH 9.5) was added (100/~l/well) and incubated at room temperature. Optical density (405 nm) of solution in wells was read after 20-min incubation. In the IDB assay, 10 /~g of test D N A was transferred to a nitrocellulose membrane. The membrane was washed in Tris-buffered saline (TBS) for 10 min and incubated in 3% gelatin in TBS for 1 h at room temperature (RT). After washing the membrane twice (5 min each) in 0.05% Tween in TBS, it was incubated in 1 / z g / m l of monoclonal antibody MoAb5 in a solution of 3% gelatin in T w e e n / T B S for 2 h at RT. After

Results

Smoking status, sex distribution, mean age and primary diagnoses of the study subjects are displayed in Table 1. Mean age and sex distribution between treatment groups are similar. Current smoking status between the 2 groups is the same, although there are more previous smokers (exsmokers) in the untreated group. The distribution of primary diagnoses, breast and hematologic malignancies, was also similar between groups. Acrolein-modified D N A ( a c r o l e i n : D N A ) was not detected by either the ELISA or IDB method

TABLE 1 CHARACTERISTICS AND PRIMARY DIAGNOSES OF THE STUDY SUBJECTS Group

N

Therapeutically treated

Untreated

controls a Cancer of tongue. F, female; M, male.

Sex

12

Mean age (years) 51 _+15.5

15

52+12.4

9F6M

7F 5M

Smoking status Previous Never Current Unknown

4 5 2 1

Previous 10 Never 3 Current 2

Primary diagnosis Breast 5 Hematologic 7 Other 0 Breast 7 Hematologic 7 Other a 1

96 2

TABLE 2 ACROLEIN : DNA DETECTION Detection method

Number of CP-treated patients (positive/ tested)

Number of untreated patients (positive/ tested)

.~

ELISA

4/12

0/15

'~

IDB

6/12

0/15

--e~----*---i---~

I

pt, I PL. 2

Pt. 3 Pt. 4

o ,u

o

ELISA. enzyme-linked immunosorbent assay: IDB, immunodot blot: CP, cyclophosphamide.

in t h e u n t r e a t e d c o n t r o l g r o u p . A m o n g t r e a t e d patients, 4/12 (33%) were positive for acrolein: D N A b y E L I S A a n d 6 / 1 2 (50%) w e r e p o s i t i v e b y t h e I D B m e t h o d ( T a b l e 2). T h e p r o p o r t i o n o f treated patients with detectable acrolein:DNA was significantly different from untreated patients u s i n g F i s h e r ' s e x a c t test ( p = 0.028, E L I S A , p = 0.003, I D B ) . A m o n g t h e 4 s a m p l e s p o s i t i v e in t h e ELISA, DNAs from patients Nos. 1-3 were much more reactive with acrolein: DNA antibody than w a s D N A f r o m p a t i e n t N o . 4 (Fig. 2) i n d i c a t i n g a

2.5

1.25

,625

.312

.156

.078

Test DNA (;ag) per well Fig. 2. ELISA optical density (405 nm) readings for DNA from 4 patients with detectable binding of acrolein : DNA antibody. Details of assay in Methods. Pt., patient,

h i g h e r level o f a c r o l e i n m o d i f i c a t i o n in t h e D N A from these 3 patients. Details of treatment history and acrolein : DNA f o r m a t i o n i n t h e p a t i e n t g r o u p a r e s h o w n in T a b l e 3. W e c o m p a r e d t h e 6 p a t i e n t s w h o d e v e l o p e d acrolein:DNA with the 6 patients who did not (using the IDB method). Using the Wilcoxon r a n k - s u m test, t o t a l d r u g d o s e , C P d o s e in last month and time since last dose did not differ

TABLE 3 TREATMENT HISTORY AND ACROLEIN : DNA FORMATION IN TREATED PATIENTS Treated patient No.

Total lifetime CP (g)

Time since last dose (months)

Smoking status ~

IDB

ELISA '~

0.5

N

+

>

1.1 11 0 0 3.0 h 0 0 11.8

P (" N N N N P P

+ + + + + -

1

12.7

2 3 4 5 6 7 8 9

9.0 7.4 2.8 5.9 13.2 7.0 14.3 0.8

1 > 48 48 5 0 48 48 1

]0

8.0

> 48

0

('

1]

3.3 12.2

36 0

0

P

1.5 b

U

0

0

0

,I

12 Untreated controls (n = 15) " ~ " a

1

Dose last month (g)

OD4o5 in wells with 5 p,g test DNA. CP orally/all others i.v. Smoking status: C, current: P, previous: N, never, U, unknown. See Table 1.

2.0 > 2.0 1.8 0.3 0 0 0 0 0 0

-

0

0

all -

all (7)

97

between the groups. However, there was a trend toward a lower white blood cell count among the patients with acrolein : D N A (z = - 1.9215, p = 0.055 corrected for ties). Discussion

Acrolein-modified DNA was detected in human peripheral blood lymphocytes from cancer patients previously treated with the anti-cancer drug cyclophosphamide. The percentage of treated patients with detectable acrolein : D N A was in the range reported by others studying cis-diamminedichloroplatinum(II)-DNA adducts (Poirier et al., 1987; Reed et al., 1986). The fact that none of the u n t r e a t e d cancer patients had detectable a c r o l e i n : D N A indicates excellent specificity of the assay. The IDB assay detected 6 of 12 CPtreated patients, whereas ELISA detected only 4 of 12 patients. This difference may be due to the higher inherent sensitivity of the IDB and the fact that more DNA (10/~g vs. 5/~g) was used in IDB than in ELISA. However, among the treated patients, there was no clear association between total drug dose and acrolein : D N A formation. There was no statistical difference in drug dose between the 6 patients with detectable a c r o l e i n : D N A who had doses ranging from 2.8 to 13.2 g (mean 8.6 g) and those with no detectable DNA damage whose doses ranged from 0.8 to 14.3 g (mean 6.6 g) (z = - 0 . 3 2 ; p = 0.75). Other authors have reported observing a partial dose response for adduct formation and cisplatin drug dose or cycles of treatment (Poirier et al., 1985; Reed et al., 1986). However this phenomenon was seen only in some patients on relatively shorter courses of cisplatin therapy (2128 days) and did not include the 30-40% of patients who never formed adducts, regardless of therapeutic dose. Differential responses to cyclophosphamide may be due to interindividual variation among patients in metabolism (activation or deactivation), adduct formation, or adduct repair (Harris, 1989; Fichtinger-Schepman, 1987b). To determine the importance of timing of exposure, drug dose in the last month was also examined as a predictor of acrolein : D N A formation. 3 of the 6 patients with detectable

a c r o l e i n : D N A by IDB had received CP during the previous month, and 3 had not. In the patients with no detectable acrolein : DNA, 2 subjects had received CP in the previous month. These data do not support the hypothesis that more recent drug exposure is associated with acrolein : DNA formation. In fact, 2 of the 6 patients with detectable acrolein: D N A had very remote previous CP exposure, i.e. 4 years or more prior to this study. This implies (i) persistence of some acrolein : D N A adducts for years, or (ii) other current sources of acrolein exposure. One of these 2 patients was a current smoker and acrolein is present in cigarette smoke (IARC, 1979); however, none of the 3 other current smokers formed acrolein : DNA. Other investigators have examined the persistence of cisplatin-DNA adducts in the blood cells. Although cisplatin does not require metabolic activation as does CP, a comparison between the persistence of D N A adducts caused by the 2 chemotherapeutic agents was of interest. Patients whose blood was drawn 24 h after the last cisplatin dose, failed to accumulate adducts when on 56-day cycles of chemotherapy unlike those on shorter 28-day cycles, suggesting that adducts were moderately long-lived with a half-life of 28 days but were removed during the longer 56-day cycle (Poirier et al., 1985). Autopsy specimens showed relatively constant levels of adducts in several different tissue samples studied 2 - 6 weeks after the last chemotherapy dose (Poirier et al., 1987). In contrast, Fichtinger-Schepman et al. (1987a) studied patients during and immediately after cisplatin infusion and found a substantial number of adducts immediately after a 3-h infusion. However, adduct removal was relatively rapid (half-life of less than 24 h) and no simple accumulation of adducts was occurring. In our study, bloods were usually sampled at least 1 month after the last CP dose with the exception of 2 patients on daily oral CP (Table 3). Thus the time between last treatment and blood collection in the 2 studies cited above and ours are different, varying from 3 h (Fichtinger-Schepman et al., 1987a) to 18-24 h (Poirier et al., 1985) to months or years (our study). This relatively long delay between treatment and blood collection in our study may contribute to the low sensitivity (6/12) of our assay in the treated group.

98

The effect of combination chemotherapy and drug dose on adduct formation has been suggested (Reed et al., 1986; Fichtinger-Schepman et al., 1987b) as an explanation for adduct formation variability. In our study, 5 of the 6 patients with detectable a c r o l e i n : D N A were on combination therapeutic regimes as were 6 of the patients without detectable acrolein: DNA, but only 1 patient was on CP alone. These results are not sufficient to allow comment on the effect of drug regime on a c r o l e i n : D N A formation. However Fichtinger-Schepman et al. (1987a) emphasized the strong inter-individual variability regarding adduct induction which could not be attributed to differences in treatment conditions or combination chemotherapy, as all patients in the study were on the same dose of cisplatin alone. Route of CP exposure was also of concern in that patients were treated by both the intravenous (i.v.) and oral route. Of the treated patients, only 2 (patients Nos. 6 and 12) received oral therapy prior to blood draw; patient No. 12 also had received i.v. CP previously. Patient No. 6 formed acrolein:DNA, implying that the oral exposure route allows DNA modification.

Conclusion Acrolein-modified DNA has been detected in peripheral blood lymphocytes of cancer patients previously treated with the anti-cancer drug cyclophosphamide. It has been postulated that adduct presence and persistence may be related to chemotherapeutic potency of anti-cancer drugs (cisplatin) as well as to the development of treatment-induced neoplasia (second malignancy phenomenon) (Poirier et al., 1985). The evidence presented here suggests that acrolein-modified DNA is induced in some patients treated with CP, although the precise nature of the a c r o l e i n : D N A modification has not been identified. With improvements in sensitivity, this marker may prove useful in monitoring the response of individual patients to CP treatment and possibly environmental exposures to acrolein.

Acknowledgements The authors wish to thank Sheryl L. Chisholm for technical assistance, and Ms. Chandra Staten

and Ms. Kerry Corbett for skilled manuscript preparation.

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Poirier, M.C., S.H. Liou, E. Reed, P.T. Strickland and M.S. Tockman (1989) Determination of carcinogen-DNA adducts by immunoassay, J. UOEH, 11 (Suppl.), 353-371. Reed, E., S.H. Yuspa, L.A. Zwelling, R.F. Ozols and M.C. Poirier (1986) Quantitation of cis-diamminedichloroplatinum II (Cisplatin)-DNA intrastrand adducts in testicular and ovarian cancer patients receiving cisplatin chemotherapy, J. Clin Invest., 77, 545-550. Schmahl, D., and M.R. Habs (1983) Prevention of cyclophosphamide induced carcinogenesis of the urinary bladder of rats by administration with mesna, Can. Treat. Rev., 10 (Suppl.), 57-61. Shamsuddin, A.K., N.T. Sinopoli, K. Hemminki, R.R. Boesch and C.C. Harris (1985) Detection of benzo[a]pyrene : DNA adducts in human white blood cells, Cancer Res., 45, 669681. Shivapurkar, N.M., S.L. Chisholm, S.N. lype and P.T. Iype (1989) Monoclonal antibodies directed against acrolein: DNA adducts, Proc. Am. Assoc. Res. Cancer, 30, 124. Snedecor, G.W., and W.G. Cochran (1976) Statistical Methods, 6th Edn., Iowa State University Press, Ames, IA, pp. 172-198. Strickland, P.T., and J,M. Boyle (1984) Immunoassay of carcinogen-modified DNA, Prog. Nucleic Acids Res. Mol. Biol., 31, 1-58. Vu, V., C.C. Fenselau and O.M. Colvin (1981) Identification of three alkylated nucleotide adducts from the reaction of guanosine 5'-monophosphate with phosphoramide mustard, J. Am. Chem. Soc., 103, 7362-7364. Yuspa, S.H., and M.C. Poirier (1988) Chemical carcinogenesis: From animal models to molecular models in one decade, Adv. Cancer. Res., 50, 25-70.