Mutation Research, 283 (1992) 97-106 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

97

MUTLET 0708

Selective reactivities of isocyanates towards DNA bases and genotoxicity of methylcarbamoylation of DNA Nobuya Tamura, Kimiko Aoki and Mei-Sie Lee Department of Chemical Carcinogenesis, Michigan Cancer Foundation, 110 E. WarrenAvenue, Detroit, MI 48201, USA (Received 9 April 1991) (Revision received 22 April 1992) (Accepted 14 May 1992)

Keywords: Isocyanates; Methylcarbamoylation of DNA; Methyl isocyanate; Phenylisocyanate; Selective reactivity

Summary The reactivities of methyl isocyanate (MIC) and phenyl isocyanate (PIC) with DNA, and the genotoxicity of MIC were investigated. MIC and PIC reacted with the exocyclic amino group of deoxycytidine, deoxyadenosine and deoxyguanosine to produce carbamoylated products. The reactions of both isocyanates with deoxycytidine were 2 and 4 orders of magnitude higher than with deoxyadenosine and deoxyguanosine, respectively. To explore the genotoxicity of MIC, M13mp9 RF DNA was modified with MIC and then introduced into E. coli. The plaque-forming efficiencies of DNA decreased with increasing dose levels, and the decreases were more pronounced in Uvr endonuclease-deficient strains (uvrA, uvrB and uvrC) than in the Uvr endonuclease-proficient strain, JM103. The differences in survival in JM103 and u v r - strains suggest that the methylcarbonyl adducts can be removed by the uvr excision-repair system. Modification of M13mp9 RF DNA with MIC induced MIC-dose-related, SOS-dependent mutations in the /3-galactosidase locus. These results demonstrate the genotoxic response of MIC-modified DNA in E. coli.

Isocyanates, R(Ar)N = C -- O, are highly reactive and are extensively used in industry. They react with a variety of nucleophiles including proteins and DNA. Since the incident of MIC contamination in Bhopal, India (Goswami, 1986), in which over 2000 people were killed and 50 000 were hospitalized, many investigators have explored the genotoxic effects of this compound.

Correspondence: Dr. Mei-Sie Lee, Department of Chemical Carcinogenesis, Michigan Cancer Foundation, 110 E. Warren Avenue, Detroit, MI 48210, USA.

For example, MIC induces sister-chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of Aroclor-induced rat-liver $9 (McConnell et al., 1987). It induces pheochromocytomas of the adrenal medulla and adenomas of pancreatic acinar cells in the male F344 rat through inhalation exposure (Bucher et al., 1989). As for human genotoxicity, using a lymphocyte culture method, Goswami (1986) has shown an increase of chromosomal aberration and sister-chromatid exchanges in the MIC-exposed survivors as compared to the control population. However, con-

90;

tradictory results have been reported regarding the mutagenicity of MIC in TA100 and TA104 in the presence or absence of rat-liver $9 (McConnell et al., 1987; Meshram and Rao, 1987; Meshram and Rao, 1988; Shelby et al., 1987). The negative mutagenicity results led to suggestions that MIC has the capacity to affect chromosome structure due to modification of nuclear protein but is not a direct mutagen (Shelby et al., 1987; Segal et al., 1989). 2-Naphthyl isocyanate (NIC), an analog of MIC, is not mutagenic in Salmonella (Wang et al., 1981), probably, because insufficient amounts of the compound are available to react with the bacterial DNA due to its instability in water. However, we have demonstrated that NIC can modify bacteriophage DNA in vitro and this modification induced genotoxic responses (Tamura et al., 1990a). The advantage of using this protocol is that the carbamoylation of DNA can be ensured, thus precluding negative results due to hydrolysis of the isocyanate. The present study was carried out to explore the selective reactivities of isocyanates towards D N A bases, and the infectivity and mutagenicity of MIC-modified M13 bacteriophage DNA (Tamura et al., 1990a). Materials and methods

Materials The 2'-deoxynucleosides were from Sigma Chemical Company, St. Louis, MO. MIC [624-835] and PIC [103-71-9], with purities > 99% and > 98%, respectively, were from Aldrich Chemical R-NH-~O~.~

CH3COO I R:CH 3 II R : P h

R-NH-CO-~H N

Company, Inc., Milwaukee, WI. M13mp9 DNA was prepared after transfection into E. coli using a sample obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN (Gupta, 1988). E. coli JM103 and Uvr endonuclease-deficient strains, AB1886 (ut'rA), AB1885 (ut,rB) and AB1884 (u~'rC), were provided by Dr. L.J. Romano, Wayne State University, Detroit, MI and Dr. B.J. Bachmann, E. coli Genetic Stock Center, Yale University, New Haven, CT, respectively.

Instrumentation methods and analytical procedures TLC was performed using Whatman PE SIL G / U V silica-gel polyester sheets with CHC13: M e O H = 5 : 1 as solvent. The chromatograms were viewed under UV irradiation at 254 a n d / o r 366 nm (Ultraviolet Products Inc., San Gabriel, CA) and they were also exposed to the 12 vapor to detect any non-UV absorbing materials. The reversed-phase HPLC analyses were carried out by use of a PRP-1 column (150 × 4.1 mm, Phenomenex, Torrance, CA) at a flow rate of 1 m l / m i n using the following chromatographic systems: (a) an initial linear gradient from 10% MeCN in 0.1 M triethylammonium acetate buffer, pH 9.0, to 50% MeCN in 10 rain and a linear gradient to 100% MeCN in 5 min; (b) a linear gradient from 20% MeCN in 0.1 M triethylammonium acetate buffer, pH 9.0, to 100% MeCN in 20 min; (c) a linear gradient from 60% M e O H in H z O to 100% M e O H in 10 rain and at 100% M e O H for 5 min, then to 60% M e O H in 4 rain; (d) a linear gradient from 50% M e O H in H 2 0 to o N

CH~COO III R : C H 3 IV R : P h

CH =COO V R:CH 3 VI R : P h

Fig. 1. Structures of carbamoylated products of di-O-acetyl-deoxynucleosides. I, N4-methylcarbamoyl-3',5'-di-O-acetyl-2'-de oxycytidine; II, N4-phenylcarbamoyl-3',5'-di-O-acetyl-2'-deoxycytidine; I11, N6-methylcarbamoyl-3',5'-di-O-acetyl-2'-deoxy adenosine; IV, N6-phenylcarbamoyl-3',5'-di-O-acetyl-2'-deoxyadenosine; V, N 2 - m e t h y l c a r b a m o y l - 3 ' , 5 ' - d i - O - a c e t y l - 2 ' - d e o x y g u a n o sine; VI, N2-phenylcarbamoyl-3',5'-di-O-acetyl-2'-deoxyguanosine.

99

70% MeOH in 20 min, then to 100% MeOH in 10 min. 1H-NMR spectra were recorded in d6DMSO solutions using tetramethylsilane as a reference and DzO was added to observe the disappearance of N - H protons (300 MHz GE NMR QE300). Fast-atom-bombardment mass spectra were recorded using thioglycerol as a matrix (Kratos MS 50TC Mass Spectrometer). Elemental analyses were performed by M-H-W Laboratories, Phoenix, AZ.

was prepared by reaction of a solution of di-OAcdC in dry pyridine with 4 molar equivalents of M1C at room temperature for 16 h to give an 85% yield of product which was recrystallized from acetone-ether-hexanes to yield an analytical sample. Compound II was prepared by reaction of a solution of Di-OAc-dC in dry dimethylformamide (DMF) with 3 molar equivalents of PIC at 37°C for 2 h to give a 67% yield of product which was recrystallized from acetone-ether to yield an analytical sample. Compound III was prepared by reaction of a solution of Di-OAc-dA in dry pyridine with 60 molar equivalents of MIC at room temperature for 3 days to give an 84% yield of product which was recrystallized from acetone-ether to give an analytical sample. Compound IV was prepared by reaction of a solution of Di-OAc-dA in dry DMF with 3 molar equivalents of PIC at 37°C for 5 h to give a 62% yield of product which was recrystallized from acetone-

Synthesis of methyl- or phenyl-carbamoylated diO-acetyl-deoxynucleosides The carbamoylated di-O-acetyl-deoxynucleosides prepared are shown in Fig. 1 and their physical data are tabulated in Tables 1-3. These compounds were prepared from reactions of MIC or PIC with the 3',5'-di-O-acetyl-derivatives of the deoxynucleosides (Koole et al., 1987; Kierzek et al., 1981; Schaller et al., 1963). Compound I

TABLE 1 M E L T I N G POINTS, TLC Rf VALUES, HPLC R E T E N T I O N TIMES (R t) A N D E L E M E N T A L ANALYSES OF COMP O U N D S S H O W N IN Fig. 1 Compounds

m.p. (°C)

Rf values a

I

211-212

0.70

II

166-167

III

Empirical formula

Elemental analysis C

H

N

7.9 b

CI5H2oN407

Calcd: Found:

48.91 49.12

5.43 5.48

15.22 14.98

0.79

9.5 c

C20H22N407

Calcd: Found:

55,81 55,93

5.12 5.05

13.02 13.08

117-118

0.70

9.7 b

CI6H20N606

Calcd: Found:

48.98 49.21

5.10 5.07

21.43 21,33

IV

163-164

0.76

15.2 d

C21H22N606

Calcd: Found:

55.51 55.67

4.85 4.85

18.50 18.38

V

209-211

0.39

8.0 c

C16H20N607

Calcd: Found:

47.06 47.28

4.90 5.04

20.59 20.43

VI

n.d. f

0.58

7.6 c

C21H22N607

Calcd: Found:

53.62 n.d.

4.68 n.d.

17.87 n.d.

a b c d e f

R t (min)

C H C I 3 : M e O H = 5:1 was used as developing solvent. HPLC chromatographic system A as described in Materials and HPLC chromatographic system B as described in Materials and HPLC chromatographic system C as described in Materials and HPLC chromatographic system D as described in Materials and Not determined due to the limited quantities of pure sample.

methods was used. methods was used. methods was used. methods was used.

If)() TABLE 2 t I-t-NMR D A T A ON C O M P O U N D S S H O W N IN Fig. 1 The values are ppm down field from tetramethylsilane reference and the other assignments are in parentheses. J values are in Hz. The data were obtained at room temperature unless otherwise indicated. Compounds

H's on sugar moiety I'-H

2'-H 2

3'-H

4'-H + 5'-H 2

Acetyl groups (s)

1

6.10 (l, J = 6.9)

2.22-2.43 (m)

5.15 (b.d, J = 5.7)

4.21 (s)

2.00, 2.04

II

6.12 (t, J = 6.9)

2.27-2.48 (m)

5.15-5.22 (m)

4.23 (b.s)

2.01, 2.(15

lII

6.42 (t, J = 7.0)

2.51-2.60 (m, 1H) 3.10-3.20 (m, 1H)

5.37-5.45 (m)

4.15-4.32 (m)

1.97, 2.07

IV

6.45 (t, J = 6.9)

2.53-2.63 (m, 1H) 3.12-3.22 (m, 1H)

5.38-5.45 (m)

4.17--4.33 (m)

1.98, 2.08

V

6.17 (t, J = 7.0)

2.45-2.52 (m, 1H) 2.85-2.98 (m, 1H)

5.27 (b.d, J = 5.4)

4.12-4.26 (m)

2.00, 2.05

V1

6.19-6.28 (m)

2.45-2.60 (m, 1H) 2.90-3.10 (m, 1H)

5.28-5.33 (m)

4.15-4.30 (m)

1.97, 2.05

Values were obtained at 65°C. b The sample availability was limited with respect to both amount and purity; the signals for the other 2 N - H protons may be buried u n d e r the background noise.

hexanes to give an analytical sample. Compound V was prepared by reaction of a solution of Di-OAc-dG in dry D M F with 100 molar equivalents of MIC at room temperature for 9 days. The product, obtained in less than a 10% yield, was isolated by preparative TLC (silica gel, CHC13 : M e O H = 4 : 1) and then recrystallized from M e O H - E t 2 0 to give an analytical sample. Compound VI was prepared by reaction of a solution of Di-OAc-dG in dry DMF with 30 molar equivalents of PIC at 37°C for 2 days. The reaction mixture was triturated with a c e t o n e ether to partially remove the side products (diphenyl urea and trimer of PIC). The crude product was then isolated by silica gel column chromatography and further purified by preparative TLC (silica gel, CHC13 : M e O H = 5 : 1). The yield was < 5% as judged by a product that was ~ 93% pure; it has an impurity at 5.2 rain in HPLC profiles using chromatographic system B.

Kinetic analysis of carbamoylation of di-O-acetyldeoxynucleosides The 3',5'-di-O-acetyl derivatives of dA, dC and dG (1/xmole) were reacted with excess MIC (100 /xmoles) or PIC (20 or 100 /xmoles) in 200 pA of dry DMF at 25°C. The unreacted isocyanates were removed from samples by a stream of argon gas or quenched with H 2 0 at various time intervals. The products formed were quantified by reversed-phase H P L C analysis using a UV detector interfaced with a Nelson Analytical Data Acquisition System (Nelson Analytical, Cupertino, CA) with chromatographic systems as described in Instrumentation methods and analytical procedures. The rate constants, kobsd , w e r e calculated using the least-squares analyses of ln{0.005 M/(0.005 M - [product])} vs. time (sec) plots.

Modification of M13mp9 RF DNA with MIC Different quantities of MIC (0-128 /xg) were

101

H's on the urea moiety

H's on base moiety N-H

Me or ~b

8.65 (b.s, 1H) 9.96 (s, 1H)

2.72 (d, J = 4.2, Me)

7.05 (t, J = 7.5, 1 p - H on 4~) 7.32 (t, J = 7.8, 2 m - H ' s on ~b) 7.45 (d, J = 7.8, 2 o-H's on ~b)

6.278, 6.272 (b.d, Cs-H on Cyt) 7.90 (d, J = 7.2, C6-H on Cyt) 6.32 a (d, J = 7.5, Cs-H on Cyt) 7.87 a (d, J = 7.5, C6-H on Cyt)

10,18 (s, 1H) 11.26 (b.s, 1H)

6.44 (d, J = 6.9, Cs-H on Cyt) 7.99 (d, J = 7.2, C6-H on Cyt)

9.20 (d, J = 4.5, 1H) 9.64 (s, 1H)

2.79 (d, J = 4.5, Me)

8.52 (s, Cs-H on Ade) 8.59 (s, C2-H on Ade)

7.05 (t, J = 7.5, 1 p - H on ~b) 7.33 (t, J = 7.8, 2 m - H ' s on ~ ) 7.60 (d, J = 7.8, 2 o-H's on 4~)

10.17 (s, 1H) 11.73 (s, 1H)

8.65 (s, Cs-H on Ade) 8.67 (s, C2-H on Ade)

2.68 (d, J = 3.9, Me)

6.74 (b.s, 1H) 10.20 (s, 1H) 11.97 (s, 1H)

8.11 (s, Cs-H on Gua)

7.06 (t, J = 7.5, 1 p - H on ~b) 7.32 (t, J = 7.5, 2 m - H ' s on 40 7.47 (d, J = 7.2, 2 o-H's on ~b)

11.40 (b.s, 1H) b

7.94 (s, Cs-H on Gua)

reacted with 16/zg of DNA in 64/xl of DMF at room temperature (22-25°C) for 2 h with gentle shaking. Following the addition of 1 ml of water,

the DNA was washed using Centricon ultrafilters and the DNA was recovered by precipitation with ethanol (Tamura et al., 1990a). The integrity of

TABLE 3 F A S T - A T O M - B O M B A R D M E N T MASS S P E C T R A L D A T A ON C O M P O U N D S S H O W N IN Fig. 1 Values are m / e

n u m b e r s and the relative intensities are shown in parentheses.

Compounds

(2M + H) +

(M + H) +

(Carbamoylated base + H) ÷

(Carbamoylated base-amine + H) ÷

Carbamoylated baseisocyanate + H) ÷

I

737 (4.1)

369 (48.1)

169

(100)

138 (6,6)

112 (26.6)

II

861 (2.8)

431 (40.6)

231

(100)

138 (8,3)

112 (38.8)

III

393 (13.6)

193

(100)

162 (29.6)

136 (73.5)

IV

909 (0.9)

455 (28.3)

255

(100)

162 (22.1)

136 (71.8)

V

817 (3.6)

409 (30.5)

209

(100)

178 (10.5)

152 (20.9)

471 (18.1)

271 a (100)

178 (62.9)

152 (92.0)

VI a

-

-

The sample used for mass spectral m e a s u r e m e n t was not absolutely pure. This n u m b e r had the highest intensity among the other

m / e values due to this compound, and was used therefore, as the base peak for comparison with the other peaks of this compound.

102 TABLE 4 REACTIVIT1ES OF MIC AND PIC WITH EXOCYCL1C AMINO GROUPS OF D1-O-ACETYLDEOXYNUCLEOSIDES lsocyanate (M) ~'

Nucleoside (M) ~

kobsd sec ~b

Mean kobsd s e c - 1

Comparative reactivity of

nucleosides to each isocyanate MIC (0.5) (0.5) (0.5)

di-OAc-dC (0.005) di-OAc-dA (0.005) di-OAc-dG (0.005)

4.2x 10 -4, 4.2X 10 - 4 2.4 X 10- 6 3.2 X 10 6 < 1.3 × 10- ~ c

2.8 X 10 6 < 1.3 × 10- s

PIC

di-OAc-dC(0.005) di-OAc-dA (0.005) di-OAc-dG (0.005) di-OAc-dG (0.005)

4.1×10 3,5.4X10 3 3.6X 10 -5, 4.9X 10 5 8.9x 10 s, 8.2x 10 -8 4.5 x 10 7, 4.9 X 10- 7

4.8x10 3 4.3X 10 -5 8.6x 10 s 4.7 × 10- 7

(0.1) (0,1) (0.1) (0.5)

4.2X 10 -4

|00 0.7 < 0.003 100 0.9 0.002 0.002 d

a Molar concentration of the reagent in the reaction mixture. b The determination of kobsd was carried out in duplicate and each value is shown. c The reactivity was so low that an accurate value could not be obtained. a Since increasing the PIC concentration from 0.1 M to 0.5 M resulted in a 5-fold increase in the kob~d value, the comparative reactivity has been divided by 5.

t h e m o d i f i e d D N A was e v a l u a t e d by elect r o p h o r e s i s o n 1% a g a r o s e gel c o n t a i n i n g 0.2 / x g / m l o f e t h i d i u m b r o m i d e . T h e m o d i f i c a t i o n by M I C d i d n o t p r o d u c e D N A s t r a n d b r e a k s , i.e. t h e r a t i o of s u p e r c o i l e d to n i c k e d p l a s m i d D N A was not visually a l t e r e d .

Viability of methylcarbamoylated M13mp9 RF DNA T r a n s f o r m a t i o n s o f E. coli w e r e c a r r i e d o u t a c c o r d i n g to a p u b l i s h e d m e t h o d ( T a m u r a et al., 1990a). Briefly, D N A was m i x e d with 0.20 ml of c o m p e t e n t cell s u s p e n s i o n p r e p a r e d by 0.1 M CaC12 t r e a t m e n t a n d t h e n h o l d i n g in ice for 30 rain. A f t e r h e a t shock at 42°C for 2 min, the cells w e r e r a p i d l y (within 5 rain) p l a t e d with 2.5 ml of t o p a g a r c o n t a i n i n g 0.25 ml of an o v e r n i g h t culture o f JM103. P l a q u e f o r m a t i o n was d e t e r m i n e d following o v e r n i g h t i n c u b a t i o n at 37°C. T h e survival curves w e r e d e t e r m i n e d f r o m 2 - 3 i n d e p e n dent transformation experiments.

Mutagenesis assay R a n d o m l y m o d i f i e d D N A s or c o n t r o l D N A s w e r e i n t r o d u c e d into c o m p e t e n t JM103 cells with or w i t h o u t t h e p r i o r i n d u c t i o n o f t h e S O S system by e x p o s i n g the host cells to 254-nm U V - i r r a d i a tion at 3 2 - 3 8 J / m 2 ( G u p t a et al., 1988). A f t e r h o l d i n g in ice for 30 m i n a n d a s u b s e q u e n t h e a t shock at 42°C for 2 min, t h e cells w e r e p l a t e d

with 2.5 ml of t o p a g a r c o n t a i n i n g 0.25 ml of a JM103 o v e r n i g h t culture, a n d 2.5 mg e a c h of 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside ( X - G a l ) a n d i s o p r o p y l - / 3 - o - t h i o g a l a c t o s i d e ( I P T G ) . T h e m u t a t i o n s p r o d u c e d in the lacZ g e n e of M 1 3 m p 9 w e r e d e t e c t e d by p h e n o t y p i c c h a n g e s o f / 3 - g a l a c t o s i d a s e activity on t h e indicator p l a t e s following o v e r n i g h t i n c u b a t i o n at 37°C. N u m b e r s f r o m at least 14 d i f f e r e n t t r a n s f o r m a tion p l a t e s ( ~ 800 p l a q u e s / p l a t e ) w e r e p o o l e d to c a l c u l a t e e a c h frequency. Results and d i s c u s s i o n

Reactiuities of isocyanates towards nucleosides in DMF It is w e l l - k n o w n that i s o c y a n a t e s r e a c t with n u c l e o p h i l e s to form c a r b a m o y l a t e d p r o d u c t s ( J o n e s a n d W a r r e n , 1970; B r o w n et al., 1987). In o r d e r to m e a s u r e the relative reactivities o f isoc y a n a t e s with t h e n u c l e o p h i l i c sites in D N A , the r e a c t i o n s of M I C a n d P I C with t h e D N A b a s e s w e r e a n a l y z e d q u a n t i t a t i v e l y using di-O-acetyld e o x y n u c l e o s i d e s . T h e p r o d u c t s f o r m e d by t h e reactions of the acetylated deoxynucleosides (0.005 M ) with excess a m o u n t s o f t h e i s o c y a n a t e s (0.1 or 0.5 M ) in D M F w e r e m o n i t o r e d by H P L C . T h e r e a c t i o n s o f M I C o r P I C with d i - O A c - d C , d i - O A c - d A or d i - O A c - d G gave a single p r o d u c t

103

in each case and their structures were confirmed as N4-methylcarbamoylated or N4-phenyl carbamoylated di-OAc-dC (I and II), N6-methyl carbamoylated or N6-phenylcarbamoylated diOAc-dA (III and IV) and N2-methylcarbamoyl ated or N2-phenylcarbamoylated di-OAc-dG (V and VI) (Fig. 1) by comparison with preparative scale samples that had been characterized as described in Materials and methods. It has been reported that thymidine is resistant to carbamoylation (Jones and Warren, 1970) and our study also showed that the reaction of 3',5'-di-Oacetylthymidine with PIC gave no carbamoylated product even after incubation with a 100-fold excess PIC for over 400 h at 25°C. The reactivities of exocyclic amino groups of nucleosides with isocyanates are summarized in Table 4. Least-squares analyses of ln{0.005 M/(0.005 M - [product])} vs. time (sec) plots conformed to pseudo-first-order kinetics, which gave correlation coefficients in the range of 0.950.99. The rate constants for phenylcarbamoylation were 1-2 orders of magnitude higher than those for methylcarbamoylation. A similar trend was reported previously for the hydrolysis of various isocyanates, which showed that the rates of hydrolysis of aryl isocyanates were significantly greater than those of alkyl isocyanates (Brown, 1987). The reactivities of the nucleosides to both isocyanates were in the order di-OAc-dC >> diOAc-dA >> di-OAc-dG. Namely, the reactivity of the exocyclic amino group of di-OAc-dC towards both isocyanates is approximately 2 and 4 orders of magnitude higher than that of di-OAc-dA and di-OAc-dG, respectively. We previously reported that the reaction of NIC with DNA yielded predominantly naphthylcarbamoylated deoxycytidine and a very small amount of naphthylcarbamoylated deoxyadenosine, however, no adduction was observed with the deoxyguanosine residues of DNA (Tamura et al., 1990a). Segal et al. (1989) have shown that in aqueous solutions, MIC reacted with calf-thymus DNA to modify mainly the Na-exocyclic amino group of cytosine residues (2.0 nmoles/mg DNA), and to a much lesser extent with the N6-amino group of the adenine residues (0.3 nmoles/mg DNA). Similarly, the present study showed that MIC and PIC reacted predominantly with the

exocyclic amino group of deoxycytidine, and to a much lesser degree with the amino groups of deoxyadenosine and deoxyguanosine. In spite of the different electronic and steric effects between methyl and phenyl groups, the similar reactivities towards the nucleosides for both types of isocyanates suggests that other isocyanates may also be highly reactive with the amino group of deoxycytidine. The similar cytidine-specific reactivity (dC >> dA >> dG) of MIC in phosphate buffer (Segal et al., 1989) and in DMF shows that solvent effects are not critical factors for the reaction selectivity. Since MIC is easily hydrolyze d in aqueous solution to yield methyl amine, in the present study the modification of bacteriophage DNA was carried out in DMF. The results with MIC and nucleosides, and our previous results with NIC and DNA, suggest that the modification of DNA with MIC occurs primarily with the N 4amino group of cytosine residues.

Viability of methyl carbamoylated M13mp9 RF DNA In order to study the effect of methylcarbamoylation on DNA survival in Uvr endonuclease-proficient and -deficient strains, the modified DNA was introduced into JM103, AB1886(uvrA), AB1885(uvrB) and AB1884(uvrC) (Yamura et al., 1990a). The plaque-forming efficiencies decreased with increasing concentrations of MIC and the decreases were more pronounced in uvrstrains, uvrA, uvrB and uvrC, as compared to that in JM103 as shown in Fig. 2. The survival curves were similar in all three uvr- strains. The effect of bulky adducts on the survival of plasmids is believed to result from the reduced potential of the modified DNA to be replicated (Lutgerink et al., 1985; Lutgerink and Loman, 1984; Tamura and King, 1990b; Tang et al., 1982). In the present study, the dose-related decrease in plaque formation in E. coli strains suggests that the modification of M13mp9 RF DNA with MIC reduces the potential of the DNA template to be replicated. Since methylcarbamoylated deoxycytidine and deoxyadenosine were stable under physiological conditions, the methylearbamoyl groups introduced by MIC are likely to be directly responsible for the reduced viabilities of the modi-

104

fled DNAs. Thus, the difference in plaque-formation of modified DNA in JM103 and in the uvrstrains suggests that this lesion can be recognized and repaired by the Uvr endonuclease complex. It has been shown that bulky alkyl lesions, such as butyl and amyl adducts introduced by alkylnitrosourea or alkylnitrosoguanidine, are repaired by the Uvr endonuclease excision-repair pathway, however, excision repair is not as efficient in removing smaller alkyl adducts such as methyl and ethyl derivatives (Todd and Schendel, 1983; Chambers et al., 1988). The Uvr excision-repair complex is thought to detect DNA distortion produced by the adduct formations as a signal of the recognition of lesions (Myles and Sancar, 1989). The methylcarbamoyl residue produced by MIC, which has a similar van der Waals volume as the butyl group (Moriguchi et al., 1976), may induce a local distortion which can be recognized by this repair system. Presumably, this local distortion causes the inhibited replication by methylcarbamoylation of DNA.

Mutagenicity of methylcarbamoylated phage DNA The phage DNAs modified with different concentrations of MIC were used for transfection of JM103 cells with or without the prior induction of SOS functions by UV irradiation and the cells

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Fig. 2. Relative survival of M13mp9 R F D N A modified with MIC in E. coli. The D N A was introduced into E. coli by a CaCl 2 procedure. After heat shock the cells were rapidly plated with top agar containing an overnight culture of JM103. Plaque formation was determined following overnight incubation. E. coli strains are represented by: •, JM103; zx, uvrA; D, uvrB; O, uvrC.

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Fig. 3. Mutation frequencies of MIC-modified M13mp9 R F D N A in JMI03. The mutations produced in the lacZ gene of M13mp9 were detected by phenotypic changes in /3-galactosidase activity on indicator plates. N u m b e r s in parentheses represent the actual counts of the mutant and normal plaques. Mutation assays, with or without SOS induction, are shown by • and O, respectively.

were then screened for mutations of the marker enzyme, /3-galactosidase, on an indicator plate which contained X-Gal and IPTG (Gupta et al., 1988). As shown in Fig. 3, increasing the concentration of MIC for the modification increased the mutagenic response in a dose-related manner in the range of the dosages employed. These mutations were dependent on the prior induction of SOS functions in the host cells; the SOS induction enhanced mutations by 4-10-fold, depending on the levels of DNA modification. In SOS-induced cells, phage DNA modified by the highest dose of MIC, from which the relative plaque forming unit (PFU) frequencies in E. coli strains correspond to those of modified phage DNA bearing more than 100 analogous naphthylcarbamoylated cytidine lesions (Tamura et al., 1990a), produced a mutation frequency of 0.2%. This frequency was 4-fold higher than the spontaneous mutation frequency in SOS-induced cells and 10-fold higher than with transfections of the modified DNA in uninduced cells. MIC has been reported to be either weakly mutagenic (Meshram and Rao, 1987, 1988) or non-mutagenic (McConnell et al., 1987; Shelby et al., 1987) in Salmonella typhimurium. However, this assay system may not be appropriate for reactive chemicals such as MIC, which can be

105 destroyed in the assay medium before sufficient amounts of the compound can react with DNA. Support for the possibility that isocyanate exposure may produce systemic toxic effects comes from the recent studies that isocyanates, such as MIC, are conjugated reversibly with glutathione (reviewed in Lepkowski, 1992). Thus, M I C conjugate may circulate in the body to various organs and tissues where, in regions of low glutathione concentration, M I C might be released from the conjugate to modify macromolecules through carbamoylation. In the present study, the mutagenicity of methylcarbamoylation was tested in JM103 using MIC-modified M13mp9 R F DNA. Although the mutagenic potency of the methylcarbamoylation product could not be determined quantitatively in these experiments, because of the undefined level of modification, the present data demonstrate the mutagenic nature of methylcarbamoylation. It is interesting to note that the mutagenicity of M I C required the induction of SOS functions. The mutagenic potential of methylcarbamoylated D N A differs from that of O6-methyl guanine lesions which induce direct base-pairing errors in the replication step to produce mutations without the need for the induction of SOS-functions (Todd and Schendel, 1983; Basu and Essigmann, 1988), even though methylcarbamoylation of the 4-amino group of cytosine may also interfere with normal G : C base pairing because the amino group is involved in W a t s o n - C r i c k base pairing with the O6-position of guanine. It has also been reported that an acetyl group linked to N4-cytosine residue does not interfere with normal G : C base pairing (Singer, 1983). Thus, it is likely that the changes of basicity of N4-amino group by these acylating agents do not cause crucial interruption of basepairing. In conclusion, the introduction of the methylcarbamoyl lesion into M13mp9 R F D N A with M I C inhibited D N A replication in vivo and this lesion was shown to induce SOS-dependent mutations in E. coli. The genotoxic methylcarbamoyl lesion formed in phage D N A can be removed by the Uvr endonuclease excision repair system in E. coli. These results suggest that the methylcarbamoylation of D N A produces effects consistent with the gross distortion of the D N A rather than

direct miscoding as a consequence of the modification of a portion of the molecule directly involved in base-pairing. Since radioisotopically labeled M I C is not readily available, the MIC-adduct levels in M13mp9 RF D N A could not be determined in the present experiments, and hence, the potency of the mutagenic effects of M I C could not be established. Future experiments, using either stable isotope dilution methodologies or postlabeling techniques, will be required to address this question.

Acknowledgements We are grateful for helpful discussions with Drs. C.M. King, C.Y. Wang and Y. Kawazoe. The assistance in the preparation of this manuscript from Mrs. E. T h o m a s - W e b e r is greatly appreciated. The authors also like to thank Dr. M.B. Ksebati and Ms. M.B. Kempff from the Central Instrumentation Facility of the Comprehensive Cancer Center of Metropolitan Detroit for obtaining N M R and mass spectra, respectively. This report from the A. Alfred T a u b m a n Facility was supported by N I H grants CA-37885 and CA-45639 and an institutional grant from the United Way of Detroit.

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Gupta, P.K., M.-S. Lee and C.M. King (1988) Comparison of mutagenesis induced in single- and double-stranded M13 viral DNA by treatment with N-hydroxy-2-aminofluorene, Carcinogenesis, 9, 1337-1345. Jones, A.S., and J.H. Warren (1970) The reaction of phenyl isocyanate with purines, pyrimidines and deoxyribonucleic acid, Tetrahedron, 26, 791-794. Kierzek, R., H. Ito, R. Bhatt and K. Itakura (1981) Selective N-deacylation of N,O-protected nucleosides by zinc bromide, Tetrahedron Lett., 22, 3761-3764. Koole, L.H., H.M. Buck, J.A. Kanters and A. Schouten (1987) Molecular conformation of 2'-deoxy-3',5'-di-O-acetyl adenosine. Crystal structure and high resolution proton nuclear magnetic resonance investigations, Can. J. Chem., 65, 326-331. Lepkowski, W. (1992) Union Carbide-Bhopal saga continues as criminal proceedings begin in India, Chem. Eng., March 16, 9-13. Lutgerink, J.T., J. Retel and H. Loman (1984) Effects of adduct formation on the biological activity of single- and double-stranded q~X174 DNA, modified by N-acetoxy-Nacetyl-2-aminofluorene, Biochim. Biophys. Acta, 781, 8191. Lutgerink, J.T., J. Retel, J.G. Westra, M.C. Welling, H. Loman and E. Kriek (1985) By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded qbX174 DNA treated with N-hydroxy-2aminofluorene, Carcinogenesis, 6, 1501-1506. McConnell, E.E., J.R. Bucher, B.A. Schwetz, B.N. Gupta, M.D. Shelby, M.I. Luster, A.R. Brody, G.A. Boorman, C. Richter, M.A. Stevens and B. Adkins Jr. (1987) Toxicity of methyl isocyanate, Environ. Sci. Technol., 21, 188-193. Meshram, G.P., and K.M. Rao (1987) Mutagenic and toxic effects of methyl isocyanate (MIC) in Salmonella typhimurium, Indian J. Exp. Biol., 25, 548-550. Meshram, G.P., and K.M. Rao (1988) Mutagenicity of methyl isocyanate in the modified test conditions of Ames Salmonella/microsome liquid-preincubation procedure, Mutation Res., 204, 123-129. Moriguchi, I., Y. Kanada and K. Komatsu (1976) Van der Waals volume and the related parameters for hydropho-

bicity in structure-activity studies, Chem. Pharm. Bull., 24, 1799-1806. Myles, G.M., and A. Sancar (1989) DNA repair, Chem. Res. Toxicol., 2, 197-226. Schaller, H., G. Weimann, B. Lerch and H.G. Khorana (1963) Studies on polynucleotides, XXIV. The stepwise synthesis of specific deoxyribopolynucleotides (4). Protected derivatives of deoxyribonucleosides and new syntheses of deoxyribonucleoside-3'-phosphates, J. Am. Chem. Soc., 85, 3821-3827. Segal, A., J.J. Solomon and F. Li (1989) Isolation of methylcarbamoyl-adducts of adenine and cytosine following in vitro reaction of methyl isocyanate with calf thymus DNA, Chem.-Biol. Interact., 69, 359-372. Shelby, M.D., J.W. Allen, W.J. Caspary, S. Haworth, J. Ivett, A. Kiigerman, C.A. Luke, J.M. Mason, B. Myhr, R.R. Tice, R. Valencia and E. Zeiger (1987) Results of in vitro and in vivo genetic toxicity tests on methyl isocyanate, Environ. Health Perspect., 72, 183-187. Singer, B. (1983) Mutagenic effects of nucleic acid modification and repair assessed by in vivo transcription, Basic Life Sci., 24, 1-83. Tamura, N., and C.M. King (1990b) Comparative survival of aminobiphenyl- and aminofluorene-substituted plasmid DNA in Escherichia coli Uvr endonuclease deficient strains, Carcinogenesis, 11,535-540. Tamura, N., K. Aoki and M.-S. Lee (1990a) Characterization and genotoxicity of DNA adducts caused by 2-naphthyl isocyanate, Carcinogenesis, 11, 2009-2014. Tang, M.-s., M.W. Lieberman and C.M. King (1982) ul~r genes function differently in repair of acetylaminofluorene and aminofluorene DNA adducts, Nature (London), 299, 646-648. Todd, M.L., and P.F. Schendel (1983) Repair and mutagenesis in Escherichia coli K-12 after exposure to various alkyl-nitrosoguanidines, J. Bacteriol., 156, 6-12. Wang, C.Y., E.M. Linsmaier-Bednar and M.-S. Lee (1981) Mutagenicity of the O-esters of N-acylhydroxylamines for Salmonella, Chem.-Biol. Interact., 34, 267-278. Communicated by H.S. Rosenkranz

Selective reactivities of isocyanates towards DNA bases and genotoxicity of methylcarbamoylation of DNA.

The reactivities of methyl isocyanate (MIC) and phenyl isocyanate (PIC) with DNA, and the genotoxicity of MIC were investigated. MIC and PIC reacted w...
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