Apparent absence of recombinogenic activity of nitropyrenes for yeast

116 0983) 119-127 Elsevier BiomedicalPress

Mutation Research,

119

Apparent absence of recombinogenic activity of nitropyrenes for yeast Elena C. McCoy a, Monika Anders a, Herbert S. Rosenkranz a and Robert Mermelstein b a Department of Epidemiology/Community Health and Center for the Environmental Health Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 and b Joseph C. Wilson Center for Technology, Xerox Corporation, Rochester, N Y 14644 (U.S.A.)

(Received 16 September 1981) (Revision received25 June 1982) (Accepted 9 July 1982)

Summaff Nitropyrenes have been shown to be potent bacterial and mammalian mutagens. However, they failed to induce any recombinogenic activity in S a c c h a r o m y c e s c e r e v i s i a e D4 even at elevated concentrations and following extended periods of exposure. A plausible explanation for this lack of activity is the absence or the lack of activation of the enzyme required for the activation of nitropyrenes in this test system under the experimental (aerobic) conditions employed.

Nitropyrenes, as well as other nitroarenes, have been recognized as potent mammalian (Nakayasu et al., 1982; see also Rosenkranz and Mermelstein, 1983) and bacterial mutagens inducing frameshift mutations in Salmonella in the absence of added microsomes (L6froth et al., 1980; McCoy et al., 1980c; Mermelstein et al., 1981, 1982; Rosenkranz and Mermelstein, 1983; Nilsson et al., 1981; Pitts et al., 1978; Rosenkranz et al., 1980; Tokiwa et al., 1981b). Although nitropyrenes can therefore, be considered direct-acting mutagens, it has been demonstrated that reduction of the nitro function to the corresponding arylhydroxylamine is required for expression of mutagenic potential (Mermelstein et al., 1982; Rosenkranz and Mermelstein, 1983; Rosenkranz et al., 1982). This appears to be accomplished by a bacterial nitroreductase that is distinct from the 'classical' nitroreductase which catalyzes the conversion of nitrofurans, nitroimidazoles and simple nitroarenes (e.g., nitronaphthalenes, nitrofluorenes) to mutagens (McCoy et al., 1981b; Mermelstein et al., 1982; Rosenkranz et al., 1981). The precise role and function of the various reductases remain to be elucidated. 0165-1218/83/0000-0000/$03.00 © ElsevierBiomedicalPress

120

Recently it was recognized that nitroarenes are widely distributed in the environment (for literature citations see Mermelstein et al., 1982; Rosenkranz and Mermelstein, 1983) as a result of the facile nitration by oxides of nitrogen of the products of incomplete combustion processes, e.g. polycyclic aromatic hydrocarbons (Pitts et al., 1978; Tokiwa et al., 1981a). Indeed, nitroarenes may be the major mutagenic species found in Diesel emissions (Claxton and Barnes, 1981; Huisingh et al., 1979; King et al., 1981; L0froth, 1981; Ohnishi et al., 1980; Pederson and Siak, 1981; Rappaport et al., 1980; Rosenkranz, 1982; Schuetzle et al., 1981; U.S. Environmental Protection Agency, 1981). Whether these nitrated chemicals are also responsible for the carcinogenicity of diesel emissions (Nesnow et al., 1982) remains to be investigated, although the carcinogenicity of l-nitropyrene was reported (Ohgaki et al., 1982). Obviously, in view of their widespread distribution and potent mutagenicity, the determination of the spectrum of genetic effects caused by nitroarenes would be of substantial interest. Such responses are of importance for a number of reasons: (a) diesel gaseous and particulate emissions are projected to increase dramatically (Ingalls and Bradow, 1981; Huisingh et al., 1979); (b) unlike the other potent microbial mutagens, exposure to nitroarenes is not restricted to humans but includes animals and plants as well. Hence they may be of ecological importance especially to the preservation of the germ plasm of highly inbred strains of cereals. This is in contrast to the protein pyrolysates Trp-2 and IQ (Kasai et al., 1980; Nagao et al., 1977, 1981; Sugimura, 1978; Sugimura and Nagao, 1979; Sugimura et al., 1977) which are combustion products present in certain foods and which are potent (indirect) mutagens but represent a risk only to those preparing or ingesting them. The present study is concerned with the ability of nitropyrenes to induce mitotic recombinations in the yeast Saccharornyces cerevisiae. This test system can supply information not only as to sensitivity to the genetic effects of this group of chemicals but it has also been suggested (de Serres, 1979) that the genetic endpoints used might also permit an estimation of hazard to the developing fetus. Deleterious recessive genes can be expressed as the result of recombinogenic events between homologous genes which would lead to lethal sectoring or malformations. In the present report it is shown that nitropyrenes, although potent bacterial mutagens, are devoid of recombinogenic activity for Saccharornyces cerevisiae when tested under standard (i.e. aerobic) conditions. These results should be contrasted, however, to those of Wilcox et al. (1982) who found that dinitropyrenes could be recombinogenic for another strain of yeast (JD1) when exposure took place under reduced oxygen tension.

Materials

The nitropyrenes used for this study were purified by HPLC and were at least 99% pure (Mermelstein et al., 1981).

121 Results and discussion

Strain D4 (Trp locus) of Saccharomyces cerevisiae was selected as the test system (Zimmermann, 1975). Exposure of the tester strain even to elevated levels of pyrene or nitropyrenes either in suspension (Zimmermann, 1975) or in plates (Brusick, 1980; Jagannath et al., 1981) did not result in the demonstration of recombinogenic activity (Tables 1 and 2). Occasionally, 1,3,6-trinitropyrene gave borderline positive results but this was not a reproducible finding. In contrast, the control 4-nitroquinoline-1-oxide (4-NQO) consistently exhibited potent recombinogenic activity (see also de Serres and Hoffman, 1981; Nagao and Sugimura, 1976; Prakash and Sherman, 1974; Prakash et al., 1974). Two plausible explanations for the lack of recombinogenic activity of nitropyrenes can be excluded: (1) Recently, it was demonstrated (Mayer and Goin, 1980) that the genetic activity of nitrophenylenediamines for Saccharomyces cerevisiae required prolonged incubation. However, a series of experiments in which exposure was prolonged for 72 h failed to reveal the genetic activity of nitropyrenes (results not shown). (2) Nitropyrene can be expected to penetrate through the cell wall of yeast, because the even larger benzo[a]pyrene moiety is capable of entry (Jagannath et al., 1981; Zimmermann and Scheel, 1981). It has been assumed that the mutagenicity of 4-NQO is dependent upon reduction of the nitro function to the corresponding hydroxylamine (see McCoy et al., 1981a). It may be of interest that Saccharomyces cerevisiae is also sensitive to the genetic activity of nitrofurans (e.g., AF-2) (Mohn et al., 1979; Murthy and Sankaranarayanan, 1978; Nakai and Machida, 1974). We have previously shown that in Salmonella, the nitroreductase acting on nitrofurans is different from the enzymes which reduce 4-NQO and nitropyrenes, respectively (McCoy et al., 1981a; Mermelstein et al., 1982; Rosenkranz et al., 1981, 1982). Thus one might hypothesize that the yeast Saccharomyces cerevisiae lacks the specific enzyme required for the activation of nitropyrenes even though it possesses other enzymes capable of acting on two other classes of nitrated chemicals. Recently, Wilcox and Parry (1981) reported that 1,6- and 1,8-dinitropyrenes induced gene conversions in the yeast Saccharomyces cerevisiae JD1, but that this genetic effect and the accompanying toxicity occurred over a narrow range of concentrations (ca. 0.4-6 ~g/ml). Further studies by these investigators (Wilcox et al., 1982) revealed that this recombinogenic effect was dependent upon a lowered oxygen tension. It would seem that these recent results in effect might explain the different experimental findings. It could well be that nitrofurans and 4-nitroquinoline-1-oxide, both of which are recombinogenic in yeast and both of which require reduction of the nitro function for expression of their genetic activity, are acted upon by the equivalent of the 'classical' bacterial nitroreductase, which in Salmonella has been shown to act on nitrofurans but not on dinitropyrenes (McCoy et al., 1981b). It might be assumed further that Saccharomyces lack the dinitropyrene-specific enzyme which is present in bacteria and this would explain the lack of recombino-

50 500 50 250 500 12.5 125 500

1,3-Dinitropyrene

1,6-Dinitropyrene

500 1000

Pyrene

l-Nitropyrene

-

p,g/ml

Solvent

Chemical

1,7 (100) 2.0 (89) 2.4 (89)

2.0 (105) 2.2 ( 9 6 ) 2.2 (96)

1.7 (88) 2.0 (64)

2.0 (94) 1.9 (84)

1.7 (100)

Expt. 1

1.9 (100)

1.6 (100)

2.4 (85)

1.4 (100) 1.1 (102)

1.7 (100)

Expt. II

Conversion frequencyX 10 5

G E N E CONVERSION I N D U C E D BY N I T R O P Y R E N E S IN Saccharomyces cerevisiae D4 a

TABLE 1

2.0(1~

Expt, II1

3.2 (100)

Expt. IV

37.0 (107)

102.0 (74)

1.8 (107) 1.9 (106)

1.5 ( 100) 1.4 (100)

1.4 (103) 1.4 (105) 1.1 (100) 1.6 (96) 1.5 (91) 1.8 (100) 1.7 (100) 1.6 (100)

48.5 (54) 17.9 (100)

18.4 (100)

18.9(100)

3.1 (88) 2.3 (65) 32.7 (69)

2.2 (88)

4.1(100) 3.3 (95) 2.8(100)

56.7 (76)

2.8(100) 2.4 (91)

1.4 (95)

Determined by the procedure of Zimmermann (1975). The numbers in parentheses indicate the extent of cellular survival expressed in per cent.

0.01

Ethyl methanesulfonate

a

0.01

4-Nitroquinoline-l-oxide

167 500

1000

17 50 100 500

1,3,6-Trinitropyrene

1,3,6,8-Tetranitropyrene

0.000125 0.00125 0.0125 0.125 1.25 12.5 125 500

1,8-Dinitropyrene

124 TABLE 2 GENE CONVERSION INDUCED BY NITROPYRENES IN Saccharomyces cerevisiae D4 a Chemical

/tg/plate

Convertants per plate Expt. I

None

Expt. 11

Expt. 11I 49

0

33

50

Pyrene

500 750

36 27

41 42

1-Nitropyrene

100 333

26 37 33

56 56 46

54

44 46 44

48

43 47

37

1000

43 45 33

41 53 62

100 500 1 000

45 45 44

48

100 333 500

72 63

111 126

135 102 I 11

104

60

147

34 38 37

56 49

51

1000

1,3-Dinitropyrene

100 500 1000

1,6-Dinitropyrene

1,8-Dinitropyrene

1,3,6-Trinitropyrene

100 500

1000

1,3,6,8-Tetranitropyrene

100 333 1000

41

4-Nitroquinoline-1-oxide

0.3

609

1275

736

Ethyl methanesulfonate

2.4

1072

1703

1347

Plate-incorporation procedure (Brusick, 1980).

genic activity of n i t r o p y r e n e s for yeast. O n the other hand, b o t h bacteria a n d eukaryotes possess oxygen-sensitive nitroreductases, some of which are rather n o n specific (e.g., x a n t h i n e oxidase). The activation of such nitroreductases u p o n o x y g e n - d e p l e t i o n could be responsible for the genotoxicity of the dinitropyrenes. I n view of the d e m o n s t r a t e d oxygen-lability of a r y l h y d r o x y l a m i n e s ( R o s e n k r a n z a n d Mermelstein, 1983), such anaerobiosis would actually be synergistic with the oxygen-labile enzymes. Recently it was shown, in fact, that while 1-nitropyrene was c a p a b l e of i n d u c i n g oncogenic t r a n s f o r m a t i o n s in cultured h u m a n fibroblasts when i n c u b a t i o n was anaerobic, this activity was abolished in the presence of oxygen ( H o w a r d et al., 1982). It would thus seem that knowledge of the p O 2 of the putative target tissue or o r g a n will be of i m p o r t a n c e in evaluating the possible consequences of exposure to nitroarenes.

125 The present findings attest further to the u n u s u a l genetic properties of n i t r o p y r e n e s a n d suggest that a n evaluation of their risk to the e n v i r o n m e n t will n o t b e a n easy matter.

Note Added in Proof The reader's a t t e n t i o n is d r a w n to a discrepancy between the values reported in T a b l e 1 a n d those reported b y us previously (Mermelstein et al., 1982; R o s e n k r a n z et al., 1982). A l t h o u g h in each i n s t a n c e nitropyrenes are reported as n o n - r e c o m b i n o genie, in the previous reports the frequency of conversions was too low by a factor of 100 due to a c o m p u t a t i o n a l error.

Acknowledgements This investigation was supported b y the N a t i o n a l Institute for E n v i r o n m e n t a l H e a l t h Sciences a n d the U.S. E n v i r o n m e n t a l Protection Agency.

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