Mutagenicity of some monoaromatic hydroxamic acids

Toxicology

Letters,

24 (1985) 111-l

111

16

Elsevier

TOXLett.

1348

MUTAGENICITY

OF SOME

(Hydroxamic

acids;

C.I.

COHEN,

WEI,

M.D.

MONOAROMATIC

HYDROXAMIC

Ames Salmonella/microsome

D.D.

Food Science and Human

Nutrition

(Received

July 5th, 1984)

(Accepted

August

27th,

SWARTZ,

assay)

S.Y. FERNANDO

Department,

ACIDS

University

and M.D.

of Florida,

CORBETT

Gainesville,

FL 32611 (U.S.A.)

1984)

SUMMARY The mutagenicity

of some monoaromatic

rat liver S-9 with SalmoneNa substituted latter

hydroxamic

derived displayed

to both strains

acids was mutagenic

hydroxamic

mutagenicity

activity,

upon

to either strain,

acids were inactive,

significant

acids was tested in the presence

and absence

of

tester strains TA98 and TAlOO. Of the five N-(chlorophenyl)-

acids and seven N-arylformohydroxamic

series were mutagenic

hydroxamic

hydroxamic

typhimurium

whereas

a consideration

metabolic

acids tested, activation.

even upon activation. the same aromatic of the nature

2 of the first and 4 of the

None of the four N-acetyl-type Because

nucleus

some of the N-acetyl-

possessing

of the aryl group

a formyl

group

in hydroxamic

acid

is important.

INTRODUCTION

Hydroxamic acids, R(Ar)CONHOH, are the N-acylated derivatives of hydroxylamine. Many of them have been shown to be carcinogenic [l, 21. Wang [3] and Wang et al. [4] also showed that some hydroxamic acids are direct-acting mutagens for S. typhimurium. Because of the applications of hydroxamic acids for agricultural, industrial and pharmaceutical uses [3], it is imperative to better understand the biological significance of these compounds. The present study investigated the structure-mutagenic activity relationships of some N-(bchlorophenyl)substituted hydroxamic acids (compounds I-.5), Nary1 formohydroxamic acids (compounds 1 and &II), and N-acetyl-type hydroxamic acids (compounds 2 and 12-14) in the Ames Salmonella assay.

Abbreviation:

DMSO,

0378-4274/85/S

03.30

dimethylsulfoxide

0 Elsevier

Science

Publishers

B.V.

112

MATERIALS

AND METHODS

Mutagenicity was tested by the plate incorporation assay of the Salmonella/ microsome mutagenicity test described by Ames et al. [5]. Strains TA98 and TAlOO were obtained from Dr. Bruce N. Ames, University of California at Berkeley. The test compounds dissolved in were spectrophotometric-grade DMSO (Schwarz/Mann, Orangeburg, NY) at 100 mg/ml and serially diluted with DMSO to 40, 20 and 2 mg/ml. Samples dissolved in 25 ,LL~ of DMSO were tested in the presence and absence of rat liver S-9 preparation from male Sprague-Dawley rats pretreated with Aroclor 1254 [.5]. Concurrent positive controls were included in all experiments; 2-aminofluorene was used for all strains in the presence of S-9, 2-nitrofluorene was used in the absence of S-9 for TA98, and methyl methanesulfonate was used for strain TAlOO in the absence of S-9. The protocol complied with the workshop recommendation on the Ames Salmonella assay [6] regarding basic techniques, such as strain maintenance, genotypic characteristic checks, S-9 preparation, and background lawn examination for toxicity. 2-Aminofluorene, 2-nitrofluorene, and methyl methanesulfonate were obtained from Aldrich Chemical Co., Milwaukee, WI. The hydroxamic acids 2, 4 and 12-14 were prepared from the corresponding Smissman and Corbett [7]. Compounds ylamines by special methods previously were prepared by the nitroso-glyoxylate RESULTS

arylhydroxylamines by the method of 3 and 5 were prepared from the hydroxdescribed [8, 91. Compounds 1 and 6-11 reaction [lo].

AND DISCUSSION

The mutagenic activity of the 4-chlorophenyl series of compounds with varying acyl groups is shown in Table I. Compounds I, 4 and 5 were mutagenic to TA98 upon metabolic activation, while compounds 2 and 3 were not mutagenic at the dose

N-C-R'

I:

6:

R’=H

“9 &

“P 8 N-C-H

“? B

R”

= H

12;

R”’ = 4-& R”’ :

4-CH,

R”‘=

4-H

2:

R’=

CH3

7:

R” = 4-Br

13:

3:

R’=

CH20H

8:

R*

14 I

4:

R’ = CH(CH,)Z

9:

R” = 3-Cl

5:

R’ = CH,CH,CO,H

Fig.

1. Structures

acids (I and 6-II),

= 4-F

IO:

R” = 4-CH,

II:

R’

= 4-C02CzH,

of N-(4-chlorophenyl) and N-acetyl-type

substituted hydroxamic

hydroxamic

acids (I-5).

acids (2 and 12-14).

N-aryl

formohydroxamic

113

TABLE

1

MUTAGENICITY TURES

OF N-(4.CHLOROPHENYL)

1-5) IN SALMONELLA

SUBSTITUTED

TYPHIMURIUM

Total revertant

Compound 1

2

3

4

5

DMSO

Dose

TAIOO

(&pIate)

- (S-9)

HYDROXAMIC

ACIDS

(STRUC-

TAIOO AND TA98 colonies/plate” TA98 + (S-9)

- (S-9)

+ (S-9)

50

98+

8

9

19k

4

32+10

500

124+

12

365 + 55

12+

3

72,

1000

92f

11

473t71

8 4

167+

8

122+21

50

109rt

14

134+ 14

21+ 21*

32+

5

500

102+

11

163 + 26

14zk 2

31+

5

1000

91+

11

163+10

16k

4

4lf

2

50

87+

14

109+25

24k

5

40+

7

500 1000

9Ok

15

162-e II

23k

4

38k

1

114*

11

178+-

31+ 10

32+

6

26k

7

47+

6

24+

4

35*

7

16+

2

40+ 11

1

9

2500

116k

17

237+21

50

105t

21

116k

500

95+

24

IlIk16

17

121k16

lo+

136k25

Toxic

1000

92k

2500

Toxic

7

64+

4

17Ok23

50

112+

10

133+ 17

33k

8

46+

500

112+

13

227 + 17

28k

8

200521

1000

117+

18

280*

19

33+

6

278 -t 33

llO+

6

136k

3

29+

8

33*

control

7

9

(25 al/plate) aMean

k S.D. from quadruplicate

levels tested. Compounds of liver S-9 mix. Although crease in revertant colony

plates.

I and 5 were mutagenic to strain TAlOO in the presence compound 3, upon metabolic activation, showed an innumbers, it was not considered mutagenic. Thus, varia-

tion of the acyl group in the 4-chlorophenyl series of compounds altered their mutagenic activity toward these S. typhimurium tester strains. It is significant that the N-acetyl derived hydroxamic acid 2 was inactive, whereas the same aromatic nucleus possessing a formyl group (I) displayed significant activity. Although we can not propose a mechanism for bioactivation from these data alone, Beland et al. [l l] have reported substantial differences between the rates of acyltransferasecatalyzed DNA binding by N-formyl, N-acetyl and N-propionyl-substituted Nhydroxy-2-aminofluorene. The relative rates of bioactivation were found to be highly species-dependent. The mutagenic activities of substituted formohydroxamic acids are shown in Table II. Compounds I, 7,8, and 10 were mutagenic in both TA98 and TAlOO upon metabolic activation. Compounds 6, 9 and ZZ were not mutagenic at the dose levels tested even with metabolic activation. It is apparent that ring substitution from

114

TABLE

II

MUTAGENICITY SALMONELLA

OF N-ARYL TYPHIMURIUM

FORMOHYDROXAMIC

Total revertant

Compound

6 7

8

TAlOO

(&plate)

- (S-9)

+ (S-9)

50

1052 12

261*

9

500

102+ IO

310+

17

11

I AND 6-11) IN

TA98 -(S-9) 36+

+ (S-9) 8

36+

I

50+ 14

118k

4

50

93+11

134+

I

36+

5

500

116kl4

195*

22

58k

7

36+ 10

50

118+ 11

278k

14

48+

I

44i

2

500

109* 10 124+ 10

324k

14

237k

3

34* 37+-

4 5

83+ 118k

9 9

105*10

284+

26

39k

6

245 i 35

50

108t

12

136+

I

42k

3

45i

500

143+

3

162+

8

42k

3

53*

4

50

15Ok

5

658+

35

45k

2

95i

IO

500

143t

9

948 zk 107

35+

3

124i

16

50

83+

6

125?

4

39k 14

44i

8

93* 12

136?

6

40+

3

44i

6

104k

17

38+- 6

4lk

6

50

10

(STRUCTURES

colonies/plate”

Dose

500 9

ACIDS

TAlOO AND TA98

500

44rt 11

9

DMSO control (25 J/plate) aMean

+ S.D. from quadruplicate

114*

4

plates.

hydroxamic acids has a strong effect on the mutagenic activity. Movement of the Cl substituent from the para to the meta position (compound I vs. compound 9) markedly depressed the mutagenic activity. The mutagenic activity of compound 10 was the strongest of all the hydroxamic acids tested. The mutagenic activity of N-acetyl-type hydroxamic acids (compounds 2 and 12-14) is shown in Table III. None of these compounds was mutagenic toward TA98 or TAlOO at the dose levels tested even upon the addition of rat liver activation system. Thus, ring substitution from N-acetyl-type hydroxamic acids has no effect on the mutagenic activity. Like the relationship between compounds 2 and 1, a comparison between compounds 7 and 12 or between 10 and I3 again showed that the N-acetyl-type derived hydroxamic acids (12 and 13) were inactive, whereas the N-formyl derivatives (7and 10) displayed significant mutagenic activity in both tester strains upon metabolic activation. This disparity in mutagenic potential between acetyl and formyl derived hydroxamic acids was previously reported in the N-acyl-N-hydroxy-2-aminofluorene series [12] and in the N-hydroxyphenacetin series [13]. There is, however, an exception to this general finding. Both compounds 6 and 14 were not mutagenic to either tester strain even upon metabolic activation. Substitution of the N-acetyl group to the N-formyl group did not affect the

115

TABLE

III

MUTAGENICITY SALMONELLA

OF N-ACETYL-TYPE TYPHIMURIUM

HYDROXAMIC

Total revertant

Compound 2

12

Dose

TAlOO

(fig/plate)

-(S-9)

+ (S-9)

- (S-9)

+ (S-9)

125 + 12

124+ 13

38+4

33 + 3

118+

9

32+3

34+4

1000

110+ 11

142+ 16

35+7

33*7

130t

8

132k 11

33f5

341-7

500

118+ 14

165? 13

36rt5

33*2

1000

118+ 14

178+ 11

31+4

34+2

50

120+

9

118+ 12

36+6

34+4

500

119&

8

121k

6

29+4

36+5

114* 10

136+

6

32k4

32+2

118+11

119+

9

33k2

32+3

500

112*11

32+3

33*3

131*11

137+ 13

36+6

33+2

117* 10

111*

33+4

33&4

118-t

1000

9

Control

(25 pl /plate) aMean

TA98

109* 10

50

DMSO

colonies/plates

50

1000 14

2 AND 12-14) IN

500 50

13

ACIDS (STRUCTURES

TAlOO AND TA98

rf-S.D. from quadruplicate

5

plates.

mutagenic activity of the N-benzo hydroxamic acids. In this case, the size of the aryl moiety may play an important role in the determination of the mutagenic activity of the hydroxamic acids as discussed by Malejka-Giganta [ 11. Our results suggest that a consideration of the nature of the acyl group in hydroxamic acid mutagenicity testing is important. Thus, data on the lack of mutagenicity of a given arylamine residue that is N-acetylated that such an arylamine type is not mutagenic. parison of the mutagenicity data for the inactive 1, 7, and 10.

should not be extrapolated to state This is best illustrated by a com2, 12 and 13 with the active analogs

ACKNOWLEDGEMENTS

This work was supported by Grant No. CA 32385 from the National Cancer Institute and by Research Career Development Award No. ES 00120 to M.D.C. from the National Institute of Environmental Health Sciences, DHHS. This is Florida Agricultural Experiment Station Journal Series. No. 5654. REFERENCES 1 M. Malejka-Giganti,

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