2-nitrofluorene and related compounds: prevalence and biological effects

Mutation Research, 196 (1988) 177-209

177

Elsevier MTR 07258

2-Nitrofluorene and related compounds: prevalence and biological effects Brita Beije 1 and L e n n a r t M/511er

2

J Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, University of Stockholm, S-106 91 Stockholm (Sweden) and 2 Department of Medical Nutrition, Huddinge University Hospital F69, S-141 86 Huddinge (Sweden) (Received 30 December 1987) (Revision received 25 April 1988) (Accepted 28 April 1988)

Keywords: 2-Nitrofluorene; Prevalence; Biological effects; Review

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Chemical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Prevalence and formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Prevalence as a result of combustion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Formation during combustion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Prevalence in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Formation in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Prevalence and formation during food processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Nitrofluorenones and nitrofluorenes other than 2NF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Summary of occurrence and formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Genotoxicity of 2-nitrofluorene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Short-term tests in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

178 178 179 179 180 181 183 184 184 185 185 185 187 187

Correspondence: Dr. B. Beije, Department of Genetic and Cellular Toxicology, WaUenberg Laboratory, University of Stockholm, S-106 91 Stockholm (Sweden).

Abbreviations: 1NP, 2AAF, 2AF, 2NF, 2NOF, 2,4,7NFone, 2,5NF, 2,7NF, 2,7NFone, 3MC, A2C r, B(a)P, CHD, CHO, GI, HDD, HPLC, i.m.,

1-nitropyrene; 2-acetylaminofluorene; 2-aminofluorene; 2-nitrofluorene; 2-nitrosofluorene; 2,4,7-t rinitrofluorenone; 2,5-dinitrofluorene; 2,7-dinitrofluorene; 2,7-dinitrofluorenone; 3-methylcholanthrene; L-azetidine-2-carboxylic acid; benzo[ a ]pyrene; Chinese hamster Don cells; Chinese hamster ovary cells; gastrointestinal tract; heavy-duty diesel; high-performance liquid chromatography; intramuscular;

i.p., LC/MS, LDD, MNNG, MN, NAC, N-OH-2AAF, OH-NF, PAH, PHS, rev, $9, SCE, SSB, SpD, TK, UDS, V79,

intraperitoneal; liquid chromatography/mass spectrometry; light-duty diesel; N-methyl-N'-nitro-N-nitrosoguanidine; micronucleated polychromatic erythrocyte; N-acetylcysteine; N-hydroxy-2-acetylaminofluorene; hydroxylated nitrofluorene; polycyclic aromatic hydrocarbons; prostaglandin H synthetase; revertants; postmitochondrial liver fraction; sister-chromatid exchange; single-strand break; Sprague-Dawley; thymidine kinase; unscheduled DNA synthesis; Chinese hamster V79 cells.

0165-1110/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

178 6.2. I n f l u e n c e o f g a s t r o i n t e s t i n a l t r a c t m i c r o f l o r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

189

6.3. G e n o t o x i c effects in v i t r o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191

Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191

F u n g u s a n d yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M a m m a l i a n cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. D N A a d d u c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. S u m m a r y o f g e n o t o x i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. I n d u c t i o n of p r e n e o p l a s t i c lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Biotrans f o r m a t i o n of 2-nitro fluorene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192 192 193 194 194 194

8.1. M a m m a l i a n m e t a b o l i s m in v i v o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. M e t a b o l i s m in isolated o r g a n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

194 196

8.3. M e t a b o l i s m in v i t r o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196

Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L i v e r fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L u n g fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prostaglandin H synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. S u m m a r y of b i o t r a n s f o r m a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. M o d u l a t o r s o f 2 - n i t r o f l u o r e n e m u t a g e n i c i t y in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196 197 197 197 197 198

10. G e n o t o x i c effects of n i t r o f l u o r e n o n e s a n d n i t r o f l u o r e n e s o t h e r t h a n 2 N F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. D i n i t r o f l u o r e n e s . . . . . . . . . . . . . . . . . . . .................................................... 10.2. 2 - N i t r o - 9 - f l u o r e n o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199 199 199

10.3. T r i n i t r o f l u o r e n o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4. 2 - N i t r o s o f l u o r e n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. M u t a g e n i c i t y of a m b i e n t air a n d diesel e x h a u s t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements ............................................................................

200 200 201 202

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

202

1. Introduction

During incomplete combustion of organic matter, nitrated polycyclic aromatic hydrocarbons (nitro-PAH) are formed, and a large quantity of different nitro-PAHs have now been identified in the environment. The aim of the present paper was to collate the work on one group of these nitro-PAHs, i.e., the nitrofluorenes. Fluorene and its nitro-derivatives are present in diesel exhaust and emission from a variety of domestic kerosene heaters and stoves. Nitrofluorenes can also be formed by photochemical reactions. Data on the prevalence and levels of nitrofluorenes in the environment are mainly available from 3 countries, West G e r m a n y (Berlin), China (Beijing) and Japan (Tokyo and Kawasaki), where mono- and di-nitrofluorenes have been identified in urban and suburban air. River sediment in J a p a n has also been shown to contain considerable amounts o f nitrofluorenes. The emphasis in this review is on 2-nitrofluorene (2NF), which is the best studied of the nitrofluorenes, and the one formed as a result of nitration

of fluorene. Its carcinogenic properties were already demonstrated in 1950. More recently a large number of studies have investigated its genotoxic activities mainly in vitro, and in some short-term in vivo studies. There is also information available on the biotransformation of 2 N F in the rat. A specific section considers the available information on the genotoxic effects of dinitrofluorenes, nitrofluorenones and 2-nitrosofluorene. 2. Chemical data

2-Nitrofluorene (2NF; CAS No. 607-57-8) consists of 2 benzene tings linked by an intermediate 5-carbon ring. 2 N F is a solid at room temperature (melting point 155-157 ° C) and has a deep orange colour. The substance has a powder-like appearance and is soluble in acetone, benzene and dimethylsulphoxide, but only sparingly soluble in water. 2 N F is easily analyzed by high-performance liquid chromatography (HPLC) and liquid c h r o m a t o g r a p h y / m a s s spectrometry ( L C / M S ) [110,111]. Since 2 N F absorbs UV light, H P L C analysis is performed with a U V detector (254-280

179 Abbreviation

Name

NO2

2NF

2-nitrofluorene

NO2

2,7NF

2,7-dinitrofluorene

NO2

2,5NF

2,5-dinitrofluorene

NO2

2NFone

2-nitro-9-fluorenone

2,7NFone

2,7-dinitro-9-fluorenone

2,5NFone

2,5-dinitro-9-fluorenone

2,4,7NFone

2,4,7-trinitro-9-fluorenone

2,4,5,7NFone

2,4,5,7-tetranitro-9-fluorenone

Structure

O

2

N

~

~ NO2

~

O

o

O2N~NO2 o

NO2 NO2 O

O2N~NO2 NO2 O

O2N~ NO2

NO2 NO2

Fig. 1. Chemical structures and abbreviations used in the text.

nm). A system with a reversed-phase column and a gradient from water to acetonitrile fulfills the analytical requirements. 2 N F has a lipophilic character in the systems described. The chemical structure of 2 N F and its related compounds is shown in Fig. 1. 3. Prevalence and formation 3.1. Prevalence as a result o f combustion

Fluorene and its derivatives are frequent byproducts of combustion, diesel exhaust being the most c o m m o n source for 2NF. Schuetzle et al. [152] have estimated the proportions of the different P A H derivatives in a diesel particulate, and they found the occurrence of fluorene derivatives to be: 34% of the P A H ketones, 18% of the P A H

carboxaldehydes, 51% of the hydroxy-PAH, 27% of the P A H quinones and 12% of the nitro-PAH. These proportions, as well as the total amounts of fluorene derivatives, vary between processes and also when light-duty diesels ( L D D ) are compared with heavy-duty diesels ( H D D ) . However, the importance of fluorene derivatives in combustion chemistry is confirmed. In the group of nitro-PAHs, 1-nitropyrene (1NP) is usually the dominating substance [19,24,38,50] but this is not always the case. Especially in H D D 2 N F may exceed 1NP by a factor of 1.8-15 with an average of 9.8 (n = 5) [38,156]. When 1NP is the dominant substance, a 2 N F concentration equal to 15% of 1 N P (n = 8) is c o m m o n (calculated f r o m [19,24,38,50]). See Table 1. A summary of 2 N F analyses in diesel exhaust is shown in Table 2. The 2 N F levels are given in

180 TABLE 1 2-NITROFLUORENE (2NF) RELATIVE TO 1-NITROPYRENE (1NP) IN DIESEL EXHAUST Vehicle

Load

Speed

HDD HDD HDD idle HDD HDD

100% 75%

Moderate High

0% 100%

High High

HDD bus cycle HDD bus cycle HDD bus cycle HDD bus cycle HDD bus cycle LDD SRM 1650 b LDD

0%

Catalyst

No cat. Cat. 2 a Cat. 3 a Cat. 4 a No cat.

2NF

1NP

2NF% of 1NP

Ref.

0.63 8.8 84 62 1.9

< 0.12/tg/g c 5.0 ~g/g c 14 ppm 3 ppm 0.13 ppm

530 180 600 2100 1500

38 38 156 156 156

0.94 0.13 0.22 0.23 0.26 4.1 15 _+1 5.52

26 /lg/krn 1.7 /~g/km 2.2 ~tg/km 3.8 ~tg/km 48 ~tg/km 43 /~g/g c 24--+3/~g/g c 27.7 /tg/g c

3.6 7.6 10 6.0 0.54 9.5 62 20

19 19 19 19 19 24 38 50

HDD, heavy-duty diesel, LDD, light-duty diesel. Bus cycle = driving cycle according to urban traffic. a Different catalysts of unknown origin, numbered by the authors of [19]. b National Bureau of Standards, reference sample. c /~g/g = ktg substance/g particulate.

/ ~ g / g particles, p p m o f p a r t i c l e s ( = / ~ g / g ) , / ~ g / k r n or / ~ g / m i l e , which m a k e s a direct c o m p a r i s o n difficult. H o w e v e r , the following c o m m e n t s c a n be made. (1) C a t a l y s t s r e d u c e 2 N F emissions b y a p p r o x i m a t e l y 80%, c o m p a r e d to n o n - c a t a l y s t s w h e n buses were tested u n d e r the s a m e c o n d i t i o n s . (2) L o w l o a d s i n c r e a s e 2 N F emission d r a m a t i c a l l y c o m p a r e d to h e a v y loads. (3) P a r t i c l e - p h a s e a n d g a s - p h a s e levels are a p p r o x i m a t e l y 5 0 / 5 0 for 2 N F . (4) 2 N F c o n c e n t r a t i o n s in diesel particles are in the r a n g e of 0 . 1 1 - 8 4 / t g / g p a r t i c l e d e p e n d i n g o n d r i v i n g c o n d i t i o n s , e.g., t y p e o f engine, load, speed. 2 N F also occurs in p e t r o l engine exhaust, alt h o u g h in low c o n c e n t r a t i o n s [50]. It is w o r t h n o t i n g that exhaust f r o m k e r o s e n e heaters c a n have c o n c e n t r a t i o n s as high as 568 n g / m 3 [107], while the c o m m o n level in u r b a n air is 0 . 0 5 - 2 n g / m 3 (see T a b l e s 3 a n d 4 for m o r e details).

3.2. Formation during combustion Air, w h i c h is n e c e s s a r y in the c o m b u s t i o n p r o cess, consists of a p p r o x i m a t e l y 80% N2, which

m a y react with 02 thus f o r m i n g various o x i d i s e d nitrogens. I n an exhaust p i p e there is a l i n e a r c o r r e l a t i o n b e t w e e n H N O 3 a n d N O 2 [204], b u t there m a y be o t h e r o x i d i s e d f o r m s of n i t r o g e n t h a t are of i m p o r t a n c e d u r i n g c o m b u s t i o n , like H2, NO~- o r H N O 2. T w o p o s s i b l e m e c h a n i s m s for n i t r o - P A H f o r m a t i o n are (a) the classical n i t r a t i o n o f P A H with H N O 3 a n d (b) the a d d i t i o n o f N O o r N O 2 to P A H free r a d i c a l s g e n e r a t e d d u r i n g c o m b u s t i o n as suggested b y Schuetzle [153]. T h e following r e a c t i o n s have b e e n suggested [147]: ( A ) N O 2 + N O + H 2 0 --~ 2 H N O 2 (B1) (2) (3) (4)

H A + H N O 3 -~ H 2 N O ~- + A H 2 N O ~- ~ N O ~ + H 2 0 NO~- + A r H --~ A r H N O ~ ArHNOf + A- ~ ArNO2 + HA

T h e r e a c t i o n p r o d u c t in A m i g h t take p a r t in r e a c t i o n B3. Scheme B is a n a c i d - c a t a l y s e d nitration of an a r o m a t i c h y d r o c a r b o n ( A r H ) . T h e levels o f NO× in the exhaust p i p e h a v e b e e n shown to b e c o r r e l a t e d with the f o r m a t i o n o f n i t r o - P A H s . W h e n the c o m b u s t i o n is m o r e c o m p l e t e , i.e., und e r o p e r a t i n g c o n d i t i o n s w h i c h increase e x h a u s t

181 TABLE 2 2 - N I T R O F L U O R E N E IN DIESEL E X H A U S T Level

Driving cycle

Comment

0.94/Lg/km 0.23/Lg/km 0.13/tg/km 0.22/~g/km 0.23/~g/km 0.26/~g/km

Bus Bus Bus Bus Bus Bus

N o catalyst Catalyst 1 a Catalyst 2 a Catalyst 3 a Catalyst 4 a N o catalyst, low load

19 19 19 19 19 19

1.5 p t g / k m 0.65/~g/km 0.11/~g/km 0.41/~g/km 0.19 t t g / k m 0.63/~g/km

Bus Bus Bus Bus Bus Bus

N o catalyst Catalyst 1 a Catalyst 2 a Catalyst 3 a Catalyst 4 a N o catalyst, low load

19 19 19 19 19 19

4.1 # g / g 15 + 1 / ~ g / g 0.63/~g/g 8.8/xg/g 5.52 /~g/g

LDD SRM 1650 c 100% load, moderate speed, H D D 75% load, high speed, H D D In muffler

24 24 24 24 50

+ b q_ b + + + + + + + +

New engine Dilution tunnel One cylinder engine, 75% load LDD, Oldsmobile 5.7 litre, V8-engine Diesel exhaust Diesel exhaust Diesel exhaust Diesel exhaust LDD Diesel exhaust

55 55 56 56 82 118 131 152 155 154

H D D , idle H D D , high speed, zero load H D D , high speed, full load Diesel particles LDD L D D gas phase (summary 1980-85) L D D particle phase (summary 1980-85) LDD

156 156 156 127 205 153 153 53

terminal terminal terminal terminal terminal terminal

FTP d

84 p p m c 62 p p m ~ 1.9 p p m c + 0.11 p p m ~ 90/~g/mile 97/~g/mile +

FTP d FTP d

Ref.

H D D , heavy-duty diesel, LDD, light-duty diesel. + , identified, not quantified. a Catalysts of u n k n o w n origin, numbered by the authors of [19]. b More than 1NP. c The National Bureau of Standards Reference Diesel Particulate. d U.S. Federal Test Procedure. Concentration in particles.

temperature centration

(increased of nitro-PAHs

speed

and

load), the con-

is significantly

with a concurrent

increase in the concentrations

partially

nitro-PAHs

oxidised

[153].

3.3. Prevalence in the environment

reduced of

Since

2NF

good reasons

occurs

in diesel exhaust

to believe that 2NF

would

there

are

be found

182 TABLE 3 FORMATION OF 2-NITROFLUORENE OTHER THAN DIESEL EXHAUST

IN

EXHAUST

Concentration

Source

Ref.

0.16 p . g / g s o o t 568 n g / m 3 19.8 n g / m 3 +

In m u f f l e r f r o m g a s o l i n e vehicle Exhaust from kerosene heater C l o s e to k e r o s e n e h e a t e r e x h a u s t Fuel aromatics + NO 2

50 107 107 55

+, identified, not quantified.

in urban air. This is indeed the case, although there are few publications concerning environmental levels. Data exist mainly from 3 countries, Japan, China and West Germany. Other publications confirm the existence of 2NF but no concentrations are given. In Table 4 the analyses of ambient air and fiver sediment are shown. The 2 N F concentrations in Japan are below 100 p g / m 3, with a higher level in Tokyo during the summer (approximately 2 times relative to winter) [62]. Beijing (China) has higher 2 N F levels in urban areas relative to suburban areas [62]. When the averages for urban and suburban areas are compared, urban areas have approximately 4 times higher concentrations. One publication covers a whole year's study of 2NF in Beijing [62] with 34 samples in total. These data have been presented in diagrams and cover a range from approximately 50 p g / m 3 to approximately 700 p g / m 3. 1NP covers the same range although the average seems to be higher compared to 2NF. In Japan the 1NP levels exceed the 2NF level in 2 cases (approximately 7 times) and in one 2NF dominates over 1NP approximately 7-fold [62]. There is a tendency for 1NP levels in Beijing to be higher in winter than in summer, whereas 2NF shows high levels during both winter and summer. In general 2 N F levels in Berlin [106,107] are higher compared to Beijing, Tokyo and Kawasaki. The average levels in Berlin are: 2.03 n g / m 3 (n = 7) October-January, area dominated by heating; 1.04 n g / m 3 (n = 7) October-January, area dominated by traffic; 1.88 n g / m 3 (n = 5) March-August, working days; 1.43 n g / m 3 (n = 5) April-September, Sundays. The range for the Berlin data is 0.17-5.22 n g / m 3. The analytical data from Berlin [106,107] have been used by the present authors to

plot the 2 N F levels over the year (Fig. 2). There are 2 series of analyses, as indicated above, a comparison between working days and Sundays from March to September, and a comparison between areas dominated by heating or traffic, covering October-January. There are no differences in 2 N F levels when working days and Sundays are compared, the compiled data forming a maximum during the period of most intense sunlight. During winter, the 2NF levels increase, especially for the samples influenced by heating. The combined data from these 2 series give a curve with 2 maxima, one during the period of most intense sunlight and the other during the coldest time of the year. The lowest levels are in early spring and autumn. Similar figures can be seen in the data from Beijing [62] although the variation is large. The lowest Beijing levels are in March and September-October with the same maxima as in the Berlin report. When comparing the 2NF levels in Berlin and Beijing with population density and traffic in the 2 cities, there are indications that the traffic might be the dominating source for 2NF in Berlin. In Beijing, on the other hand, local burning of coal for cooking and heating may be the major source of nitro-PAHs. The Japanese data are limited, and they show low levels [62]. This might be explained by the fact that (a) very few use their vehicles to go to work,

ng/m

3 654321-

,~.,.~ D m

• .',~:~.

-

0 F

M

A

M

J

J

A

S

O

N

D

J

Month

Fig. 2. 2-Nitrofluorene (2NF) levels in air samples from Berlin collected over 1 year. Air samples from March to September were collected during working days (filled squares) and Sundays (open squares). Air samples from October to January were collected from an area dominated by traffic (filled circles) and an area dominated by heating (open circles). The data have been extracted from references 107 and 108.

183 TABLE 4 2-NITROFLUORENE IN THE ENVIRONMENT Concentration

City/Country

Comments

71 p g / m 3 24 p g / m 3 50 p g / m 3

Kawasaki/Japan Tokyo/Japan Tokyo/Japan

Industrial site, close to road, autumn Urban site, residential, winter Urban site, residential, summer

62 62 62

190 p g / m 3 290 p g / m 3 36 p g / m 3 79 p g / m 3 50-700 p g / m 3

Beijing/China Beijing/China Beijing/China Beijing/China Beijing/China

Urban site, close to road Urban site, close to road Suburban site Suburban site n = 34 (over a year) a

62 62 62 62 62

+ 1.42 0.88 0.74 3.22 0.54 2.25 5.22

ng/m3 n g / m3 ng/m3 n g / m3 ng/m3 ng/m3 ng/m3

Berlin/W. Berlin/W. Berlin/W. Berlin/W. Berlin/W. Berlin/W. Berlin/W. Berlin/W.

Germany Germany Germany Germany Germany Germany Germany Germany

2NF in all samples October, area dominated by heating b November, area dominated by heating b November, area dominated by heating b December, area dominated by heating b December, area dominated by heating b January, area dominated by heating b January, area dominated by heating b

108 106 106 106 106 106 106 106

0.43 0.37 1.02 0.31 2.76 2.21 0.17

ng/m3 n g / m3 ng/m3 ng/m3 ng/m3 n g / m3 ng/m3

Berlin/W. Berlin/W. Berlin/W. Berhn/W. Berlin/W. Berlin/W. Berlin/W.

Germany Germany Germany Germany Germany Germany Germany

October, area dominated by traffic c October, area dominated by traffic c November, area dominated by traffic c December, area dominated by traffic c December, area dominated by traffic c January, area dominated by traffic ~ January, area dominated by traffic c

106 106 106 106 106 106 106

1.12 0.58 4.19 3.07 0.45

n g / m3 ng/m3 n g / m3 n g / m3 ng/m3

Berhn/W. Berhn/W. Berlin/W. Berlin/W. Berlin/W.

Germany Germany Germany Germany Germany

March, workdays d April, workdays ,l May, workdays d July, workdays a August, workdays a

107 107 107 107 107

0.38 1.68 2.87 1.20 1.00 +

n g / m3 ng/m3 ng/m3 ng/m3 ng/m3

Berlin/W. Germany Berlin/W. Germany Berlin/W. Germany Berlin/W. Germany Berlin/W. Germany Tokyo/Japan

April, Sundays e May, Sundays e July, Sundays e August, Sundays e September, Sundays e

107 107 107 107 107 150

Suimon River/Japan

Sediments

151

1.5/.tg/kg

Ref.

+ = identified, not quantified. Average: a No average calculated since all data were displayed in a diagram. b 2.03 n g / m 3 (area dominated by heating). c 1.04 n g / m 3 (area dominated by traffic). a 1.88 n g / m 3 (March-September, workdays). 1.43 n g / m 3 (March-September, Sundays).

( b ) t h e r e a r e c a t a l y s t s o n t h e v e h i c l e s , a n d (c) t h e

3.4. Formation in the environment

cities of Tokyo and Kawasaki are situated on the c o a s t . A s i n d i c a t e d i n T a b l e 4, 2 N F h a s a l s o b e e n f o u n d i n r i v e r s e d i m e n t s i n J a p a n [150].

The 2 major

sources for 2NF

in the environ-

ment are diesel exhaust and emission from various

184

TABLE 5 FORMATION OF 2-NITROFLUORENE BY PHOTOCHEMICAL REACTIONS Reaction F + nitrite+ UV light = increased mutagenicity a F + NO/= 2NF 2AF + sunlight = 2NF 2AF + fluorescent light = formation of mutagens a 2AF + UV = 2NF

Ref. 120 178 126 196

117,126,127,128, 170,171,172

2NF, 2-nitrofluorene, 2AF, 2-aminofluorene, F, fluorene, UV, ultraviolet fight. a No identification of reaction products.

heating processes. However, as indicated by data from both Berlin and Beijing, there is an increase of 2 N F during the period with most intense sunlight. This fact is in accord with the data in Table 5, where it is shown that 2NF can also be formed by photochemical reactions. Most of the studies have been performed with 2-aminofluorene (2AF) and UV light, with the formation of 2NF [117,127,128,171,172]. One study [196] showed an increase in mutagenicity, but no reaction products were identified. Two studies have used fluorene as a starting reagent. One used fluorene + nitrite and UV light with an increased mutagenicity as a result [120], the other used fluorene + NO2, which produced 2NF [178]. Furthermore, it has been shown that the formation of nitro-PAHs is enhanced by the common air pollutants SO 2 and H N O 3 [178]. As stated above, the precursors for photochemical 2 N F formation are fluorene or 2AF. Fluorene can be found in urban air at levels of 4-18 n g / m 3 [2], and also in diesel fuel [56]. 2AF can be found in oil at levels of 6 _+ 3 # g / g [1801. There might also be industrial sources of nitroPAHs since they are used as intermediates in chemical synthesis [33,136,138].

nitro-PAHs are detected in complex samples there are good reasons to believe that a large variety of nitro-PAHs exists even if chemical analyses only result in the identification of one or two nitroPAHs. Various potent mutagens are formed during certain food processing, especially cooking at high temperature [175]. It is therefore not surprising that nitro-PAHs have also been found in food after preparation over a domestic gas flame (combustion of gas), especially when food like chicken is cooked under acidic conditions (marination with sour sauce) [72]. 2NF was not analysed in this study [72], but 4 other nitro-PAHs were, among them 1NP. The nitropyrene formation was pH-dependent and resulted in levels between 0.5 and 10.7 n g / g chicken. In addition to this, the airborne mutagenicity (Salmonella TA98) in the kitchen increased 36 times during cooking. This would be relevant in Japan since this way of preparing chicken is popular. It also raises the question whether food, or at least certain cooking procedures, gives a substantial addition of nitro-PAHs. If a person eats 200 g chicken (one meal) with 10.7 ng 1 N P / g , the total dose will be 2.1 ~tg. This dose can be compared with the polluted air of Berlin where the average 1NP levels are 3.4 n g / m 3 [106]. Thus, in a theoretical calculation of exposure, when a person inhales 15 m3/day, the chicken meal corresponds to 40 days (24 h / d a y ) exposure on a heavily polluted street in Berlin. If the same comparison is made with 1NP levels in Tokyo [62] this chicken meal corresponds to 3.5 years of breathing on the streets of Tokyo. Nitro-PAHs have also been detected in peated malt and tea [36]. Grilling under an open flame (domestic gas flame) of corn, bacon, beef, pork and fish results in the formation of nitro-PAHs [122]. When assessing risk due to nitro-PAH exposure, food processing should not be neglected although the above cases could be extreme situations. 3.6. Nitrofluorenones and nitrofluorenes other than

3. 5. Prevalence and formation during food processing

2NF

2NF and 1NP occur together with other nitroPAHs, fluorenes and pyrenes being among the most common subgroups of the nitro-PAHs. When

There exist a number of fluorenes related to 2NF. The addition of 2 nitro groups leads to nitration in positions 2,5 and 2,7. Dinitrofluorenes

185

(2,5 and 2,7NF) have been identified in airborne particles from Japan [126] and in diesel particles [131]. The 2,7NF isomer was also found in river sediments in Japan [151] and in U.S.A. urban air [199]. Moreover, fluorene derivatives form a large group of substances in combustion processes (section 3.1). 9-Fluorenone is a common oxidised form of fluorene which can also be mono-nitrated by photochemical reactions [117,129,170] or dinitrated (2,7NFone) during combustion [131]. One sample of airborne particles from Japan gives the following figures [87]. pg/m 3

Per cent

1NP 2NF 2,5NF 2,7NF + 15 other nitro-PAHs

140 50 ) 190 1500 1264

4.5

In total

3144

55.3 40.2 100.0

The trinitrofluorenone, 2,4,7NFone, is a photoconducting agent, which can be present in photocopying machines. It has also been used as a fungicide and as a complexing agent in analysis of indoles by mass spectrometry [32,57,165,183]. 3. Z Summary of occurrence and formation Nitro-PAHs and nitrofluorenes are found where incomplete combustion takes place, both in the urban and suburban environment and in the domestic milieu. In addition 2NF can be formed in photochemical reactions. 2NF and its derivatives are found in the environment all over the world, in developed as well as underdeveloped countries. They represent a large part of the nitro-PAHs and occur in both the particle and the gas phase. Not only are vehicles responsible for the emission of nitro-PAH, but food can also be an important source. In risk assessment of nitroPAHs it is therefore important to take into consideration (a) the specific nitro-PAH, (b) the route of administration, and (c) the variable sources from which exposure occurs.

4. Toxicity There is little information regarding pure toxicological effects (toxicity) due to 2NF. However, a

10-15% decrease in body weight, with no major signs of toxicity, was observed in male SpragueDawley (SpD) rats treated with intraperitoneal (i.p.) injections of 224 #mole 2 N F / k g body weight, once daily for 5 days [206]. In a feeding study, no adverse effects of 2NF on survival were observed in rats (male and female) fed a diet containing 1.62 mM 2 N F / k g over an 8-month period, or until the time of tumour formation [103]. The interpretation of the condition of rats used in another feeding study [109] is somewhat obscured by the fact that a synthetic diet was used when 2NF was given in the diet. The average length of survival in control rats eating a standard diet was about twice that of those receiving the synthetic diet. All groups of rats treated with 2NF by skin painting, and being fed the standard diet, survived longer than rats ingesting the carcinogen in a synthetic diet, and also longer than control rats on the synthetic diet. In male Swiss-Webster mice (25-30 g), the 24-h LDs0 for 2NF has been shown to be 1.6 g / k g [162]. 2NF, per se, has not been tested for teratogenic effects. However, some of its possible metabolites have been shown to be teratogenic [39,40]. It is known that 2NF was non-toxic towards Salmonella in a modified Ames assay, at doses up to 333/~g/plate [98]. Neither was 2NF toxic to the fungus A. nidulans up to 2000 ~ g / m l in an incubation system [20]. In mammalian cell cultures, on the other hand, toxic effects of 2NF have been observed. Thus, a 2NF concentration of 9.48 x 10 -4 M caused 50% lethality in Chinese hamster Don (CHD) cells [48], and 400 /~g 2 N F / m l incubation system reduced the survival in mouse lymphoma cells to 16% [119]. 5. Carcinogenicity Already in 1950, Morris et al. [109] published cancer data with 2NF. Male and female rats were fed a diet containing 0.05% of various fluorene derivatives (2NF, 2AF or 2AAF) for 23 weeks, after which the surviving rats were fed the control diet until they developed tumours or death ap~ peared imminent. In a parallel study the carcinogens were applied to rats by skin painting (3 drops 3 times weekly for 6 months and thereafter 6 drops 3 times weekly for 10-15 months). The

0 3

8 7

2 1 2 1 1

1

2 3

1 1

0 6

M

9

0 2

4 0

F

9

1 5

0 0

M

20

2 17

13 1 4

M

F

M

F

4 5

4

F

5 6

3 1

1

M

F

8 6

2 5 1

1 5

1

M

9

0

0 7 5

F

oral c

9

4

9 0 5

M

Miller et al. *

11 5

1

1 5 1 1 1 1

F

4 4

1

1 2

M

painting a

Morris et al. *

7 10

1

3

3

F

oral b

2-Acetylaminofluorene

16 10

1

6 5 4

M

27

6

0 22 19

F

26

13

24 0 11

M

Miller et al. * oral c

* Data extracted from Morris et al. [109], and Miller et al. [103]. The data from Morris' study give the numbe r of tumours detected, and the d ata from Miller's study give the number of rats with tumours in the respective tissue after 10 or 12 months. F = female, M = male. a Three drops of a 2% acetone solution of the compound, 3 times weekly for 6 months, thereafter 6 drops 3 times weekly. b Synthetic diet containing 0.05% of the compound was given to the rats for 23 weeks, thereafter the synthetic control diet. c Stock grain diet containing 1.62 m M of the c o m p o u n d / k g diet was given to the rats for 8 months, thereafter the grain diet alone for 2 months. d Stock grain diet containing 1.62 m M of 2 N F / k g diet was given to the rats for 12 months.

Total tumours Total rats

Liver M am m a ry gland Ear duct Skin Pituitary gland Adrenal gland Lung Subcutaneous Salivary gland Thymus gland Kidney Bladder Intestine Forestomach

oral b

oral c

oral b

painting a

painting a

Morris et al. *

Miller et al. *

Morris et al. * oral d

2-Aminofluorene

2-Nitrofluorene

T U M O U R D I S T R I B U T I O N IN RATS EXPOSED TO 2 - N I T R O F L U O R E N E , 2 - A M I N O F L U O R E N E O R 2 - A C E T Y L A M I N O F L U O R E N E

TABLE 6

187 average survival time for rats given the carcinogens orally was 308-310 days for 2NF, 150-285 days for 2AF and 200-240 days for 2AAF. The survival time for skin-painted rats was longer, 310-560 days for the 3 carcinogens. The rats ingesting 2NF, 2AF or 2AAF developed fewer tumours than rats painted with these compounds, even though approximately 10 times more carcinogen was administered orally than by painting. However, in the painting experiments, the rats were treated over a much more extended period. N o liver tumours were induced by 2 N F in this study, whereas 2AF induced cholangiomas, and 2AAF mostly hepatomas. 2 N F induced tumours in m a m m a r y gland, ear canal, pituitary gland, adrenal gland, lung and salivary gland regions. The largest number of tumour sites was found in males after painting with 2NF. Painting with 2AAF, on the other hand, produced the largest number of tumour sites in females (liver, m a m m a r y gland, ear canal, pituitary gland, adrenal gland and intestine). H e p a t o m a s were only induced by orally administered 2AAF (both females and males). Only 2AF gave rise to skin carcinomas near the site of painting (Table 6). In a cancer study performed 5 years later by Miller et al. [103] the rats were fed a diet containing the same carcinogens, i.e., 2NF, 2AF or 2AAF, at a concentration of 1.62 m M / k g diet for 8 months, after which the rats were kept on a standard diet for a further 2 months. In agreement with Morris et al. [109] 2AF was less active than 2AAF, primarily due to an increased latency period. 2 N F was likewise much less active at the sites affected by 2AAF. However, 2 N F was the only compound causing multiple tumours in the forestomach (Table 6). To confirm this observation, Miller and coworkers performed a further study [103], in which a group of 20 male rats were fed 1.62 m M 2 N F / k g diet for 12 months. Of the 18 surviving rats, 17 had squamous-cell carcinomas in the forestomach. In no case was the glandular stomach involved. In addition 13, 4, 2 and 1 of the rats had tumours of the liver, ear duct, small intestine and m a m m a r y gland respectively at 12 months (Table 6). N o tumours were found in the 10 control rats. The absence of tumours in the forestomach of rats in the study by Morris et al. [109] is probably due to the shorter exposure time.

More detailed information about the carcinogenicity of 2 N F and its possible implications to humans will be available in a forthcoming monograph on diesel exhaust, which is in preparation at the International Agency of Research on Cancer, Lyon. Miller et al. [101] suggested 2-nitrosofluorene (2NOF) and N - O H - 2 A A F as ultimate carcinogens during activation of 2AAF, because the formation of 2 N O F from N - O H - A A F had been demonstrated in vitro with liver microsomes. However, it was not possible to verify this reaction in vivo, probably due to the high reactivity of 2 N O F [100]. Neither has it been possible to demonstrate any adduct formation with 2NOF, although N - O H 2AAF readily reacts with guanosine to form 8-(N2-fluorenylacetamido)-guanosine in an incubation mixture containing rat liver soluble enzymes [70]. Subcutaneous injections of 2.5 mg 2 N O F twice weekly for 8 weeks induced sarcomas at the injection site in 10 out of 20 female rats and m a m m a r y carcinomas in 9 out of 20 [100]. When 2 N O F (at equimolar dose to 3 mg N - O H - A A F ) was injected subcutaneously once weekly for 6 weeks, 10 out of 17 male rats had developed sarcomas at the injection sites after 14 months, whereas no tumours were detected in the ear ducts or m a m m a e [102]. Similar results were obtained with N - O H - 2 A A F , whereas no tumours at all developed within 14 months after 2AAF or 2AF injections subcutaneously. 2 N O F is further discussed in section 9.4. A single dose of 2,4,7-trinitrofluorene (2,4,7NF) caused an increase in m a m m a r y tumours in female rats [60]. 2,4,7NF is further discussed in section 10.3.

6. Genotoxicity of 2-nitrofluorene 6.1. Short-term tests in oivo

Only a few studies have dealt with the genotoxic effects of 2 N F after short-term treatment in vivo. However, these studies indicate that the route of administration may play an important role. Thus oral administration of 2 N F has generally given a positive result (except for one study using the host-mediated assay), whereas i.p. injection has given a negative result.

188 I n T a b l e 7, the different in vivo studies are s u m m a r i s e d . O n e l a b o r a t o r y [115] m e a s u r e d the i n d u c t i o n o f s i s t e r - c h r o m a t i d e x c h a n g e (SCE) in C h i n e s e h a m s t e r b o n e m a r r o w after b o t h oral a n d i.p. a d m i n i s t r a t i o n of 2 N F . W h e n 2 N F was a d m i n i s t e r e d orally a positive effect was observed, while 2 N F a d m i n i s t e r e d i.p. was negative. In a n o t h e r s t u d y using m o u s e b o n e m a r r o w , n o ind u c t i o n of m i c r o n u c l e a t e d p o l y c h r o m a t i c e r y t h r o cytes ( M N ) was observed. However, in this s t u d y o n l y the i.p. r o u t e h a d b e e n e m p l o y e d [121]. F u r thermore, 2NF did not induce sperm-head a b n o r m a l i t i e s in mice when the c o m p o u n d was a d m i n i s t e r e d i.p. [181]. F o l l o w i n g oral a d m i n i s t r a t i o n , 2 N F i n d u c e d D N A r e p a i r in rat liver, the extent o f the i n d u c tion being a p p r o x i m a t e l y half o f the effect o b served with 2 A A F at the s a m e dose (25 m g / k g b o d y weight). D N A r e p a i r was m e a s u r e d a u t o r a d i o g r a p h i c a l l y as u n s c h e d u l e d D N A synthesis ( U D S ) in h e p a t o c y t e s isolated f r o m the e x p o s e d rats. T h e i n d u c t i o n of U D S was o b s e r v e d at b o t h 12 a n d 24 h after the a d m i n i s t r a t i o n of 2 N F [15,16]. W h e n given orally, m e t a b o l i t e s of 2 N F are excreted in the u r i n e a n d faeces, giving rise to m u t a g e n i c activity when i n c u b a t e d with S a l m o n e l la TA98. T h e m a x i m a l m u t a g e n i c effect in urine

has b e e n o b s e r v e d 24 h after a d m i n i s t r a t i o n a n d in faeces 48 h after a d m i n i s t r a t i o n . T h e m a j o r d i r e c t - a c t i n g m u t a g e n i c i t y (75%) in the urine was a s s o c i a t e d with the u n c o n j u g a t e d m e t a b o l i t e s , a n d this m u t a g e n i c activity was r e d u c e d b y the a d d i t i o n of a m e t a b o l i s i n g s y s t e m (rat liver $9). M u t a g e n i c m e t a b o l i t e s in the urine have b e e n identified as N - O H - A A F a n d O H - N F s , a n d in faeces as O H - N F s a n d to s o m e e x t e n t u n m e t a b o lised N F . F o u r d a y s after the a d m i n i s t r a t i o n of 14C-2NF (25 m g / k g b o d y weight) m o r e t h a n 90% of the dose h a d been excreted [112]. W h e n 2 N F was tested in the h o s t - m e d i a t e d assay b y 2 different l a b o r a t o r i e s [162], a d u l t m a l e S w i s s - W e b s t e r mice were used as hosts a n d S a l m o n e l l a TA1538 o r S. cerevisiae D3 as test organisms. 2 N F was positive t o w a r d s T A 1 5 3 8 in o n e l a b o r a t o r y ( R o s e n k r a n z ' s ) b u t n o t in the o t h e r ( S i m m o n ' s ) . T h e different results m a y be d u e to v a r y i n g doses a n d m o d e s of a d m i n i s t r a t i o n used b y the two groups. S i m m o n a d m i n i s t e r e d the 24-h LDs0 d o s e (1600 m g / k g ) of 2 N F b y oral gavage, while R o s e n k r a n z a d m i n i s t e r e d a p p r o x i m a t e l y 1 / 1 0 of this dose i n t r a m u s c u l a r l y (i.m.) [162]. N o effect was o b s e r v e d with S. cerevisiae D3, which was o n l y used b y S i m m o n . T h e s e results are at v a r i a n c e with the positive i n d u c t i o n o f S C E a n d

TABLE 7 GENOTOXIC EFFECTS OF 2-NITROFLUORENE IN VIVO Administration route a

Dose (mg/kg) b

Species

Assay

Genotoxic activity

References

oral oral oral oral

12.5-50 125-500 1600 50

Rat Hamster Mouse Rat

Liver UDS Bone marrow SCE Host-mediated (TA1538) * Mutagenicity in urine and faeces Bone marrow SCE Bone marrow MN Sperm head abnormalities Host-mediated (TA1538)

+ + +

15, 16 115 162 15, 16

+

115 121 181 162

i.p. i.p. i.p. i.m.

50-200 12.5-200 c 250-1250 d 125

Hamster Mouse Mouse Mouse

UDS, unscheduled DNA synthesis, SCE, sister-chromatid exchange, MN, micronuclei. a oral = dosed by gavage, i.p. = intraperitoneal injection, i.m. = intramuscular injection. b mg/kg body weight as a single dose. c As one single injection or 5 daily injections. d Five daily injections. Smears of caudal sperm prepared 5 weeks after the last injection. Lethal dose 1500 mg/kg/day. S. cereoisiae D3 also gave a negative result.

189 M N in bone marrow and U D S in liver after oral administration of 2NF. The influence of possible metabolic pathways is discussed in section 8.1. An interesting comparison can be made with the results from a more widely studied nitroarene, i.e., 1NP, which is active in 2 host-mediated assays using mouse (E. coli) and rat (Salmonella). It also induces SCE as well as M N in hamster and rats following i.p. administration and causes D N A damage in mouse lung as a result of intratracheal administration [147]. 1NP gives both positive and negative results in various in vitro mutagenicity assays. The free-living soil nematode Caenorhabditis elegans has been used to study in vivo mutagenesis by reversion of small-sized animals to larger 'wild-type' animals. A strong positive effect was obtained with 2 N F [81].

6.2. lnfluence of gastrointestinal tract microflora The abundance of bacteria with nitroreductase activity in the gastrointestinal (GI) tract of normal rats and the fact that nitroreduction seems to play an important role in the metabolism of 2 N F have stimulated several investigations with germ-free rats. After a single oral dose of 2 N F (40 m g / k g body weight) to germ-free A G U S rats, urine and faeces were collected and tested for mutagenic activity towards Salmonella TA98 in the plate incorporation assay ( + rat liver $9). Both urine and methanol extracts of faeces induced mutagenicity that far exceeded the effects obtained with conventional rats of the same strain treated simultaneously [111]. The increased mutagenicity observed with the germ-free rats could be related to the presence of hydroxylated 2NFs, as will be discussed in section 8.1. A colonisation of Salmonella TA1538 within the G I tract of other,vise germ-free S p r a g u e - D a w ley (SpD) rats can be maintained for several months. The bacteria are found in the stomach, lower bowel and faeces. After feeding with 2 N F (3.4 r a g / r a t / d a y for 3 days) or 2AAF (3.6 m g / r a t / d a y for 5 days), the concentration of TA1538 mutants in faeces increased, whereas the same treatment with the 2AAF metabolite 7-OHA A F (7 m g / r a t / d a y for 5 days) did not induce

mutations in TA1538 [46,47,164,195]. A single oral dose of 2 N F (3.4 mg) to SpD rats increases the concentration of TA1538 revertants in faeces to a peak 48 h after administration of the mutagen, thereafter returning to control values within 6 days. A dose-response relationship for 2 N F ingestion and TA1538 revertants in the faeces was also demonstrated [25,47]. The addition to germ-free rats of bacteria representative of the normal gut flora diminishes the number of Salmonella revertants found in the faeces in response to the feeding of 2NF. Thus, when Lactobacillus plantarum and Bacteroides fragilis were associated together with TA1538, the revertant response to 2 N F was much lower than the one mentioned above in rats colonised only with TA1538 [47,164]. A similar effect was also observed with germ-free rats associated with TA1538 and Bacteroides oulgatus, the his + revertant response to orally administered 2 N F being very weak. The concentration of B. vulgatus in an individual was negatively correlated with the response to 2NF. The explanation given for the reduction in mutagenic response to 2 N F in the presence of B. vulgatus was the fact that B. vulgatus readily reduces 2 N F to 2AF, which is less active as a mutagen in vitro [26,46]. The Salmonella strain TA1535, colonised in germ-free rats, was not affected by 2 N F treatment, which is consistent with the lack of effect with this Salmonella strain in the plate incorporation assay [90,99,161]. Contrary to the above-mentioned in vivo study, in vitro studies with cell-free extracts from h u m a n anaerobes ( B. fragilis, B. oulgatus, B. thetaiotaomicron) have shown that extracts from all 3 bacteria are capable of converting 2AF to mutagens (1-3 revertants//~g 2AF) in the plate incorporation assay with TA1538, and B. vulgatus and B. thetaiotaomicron increase the mutagenic activity of 2 N F 2-fold compared to its direct-acting mutagenicity. B. fragilis and B. thetaiotaomicron are also capable of converting 2 N F to mutagens active for the nitroreductase-deficient Salmonella strain TA1538-FR (2.2 and 6.4 revertants//tg 2NF, respectively) [69]. Moreover, 2AF and 2-aminoanthracene are converted to mutagens by the combined action of microsomes derived from the rat intestinal mucosa and of cell-free extracts prepared from a human strain of B. fragilis, under

190 TABLE 8 GENOTOXIC

EFFECTS OF 2-NITROFLUORENE

Test organism

IN BACTERIA IN VITRO

Genetic marker

Metabolic system e

Genotoxic effect g

References

Salmonella a TA1534 TA1535 TA1537

hishishis

-

+ +

6 90, 97, 99, 161 69, 90, 97, 99, 161

TA1538

his-

-

+

7, 34, 85, 90, 97, 98, 99, 161

TA1978 TA98

hishis-,R

-

(+ ) +

7, 145 34, 85, 90, 93, 97, 99, 161

TA100 TA98NR TA98NR

his-,R his-,R his ,R

+ f

+ (+)/(+)

11, 90, 161 28, 93, 94, 144 148

TA98/1,8DNP 6 TA100FR

his ,R his-,R

-

( + )/-

93, 94, 96, 144 8

Salmonella

his-,Ara ~b

-

+

27, 149

E. coli

polApolA polA-

+ +

+ + -

148 92 42, 163 84

p3478 WP2/WPI00

polA -

-

-

W3110

polA +

+

+

92

JC5519 JC5547 JC2926 JC2921 WP100 CM6ll

recBrecArecA recAuvrAuvrA-

_+ + +

+ / + +

61,176 176 176 176 84, 85, 92 92

WP67 WP2 CM891 WP2 CM891 PQ37

uvrAuvrAuvrAtrpuvrAlexA

+ _+ + + + +

+ +/( + ) + + +

92 92, 97 104, 105 105 104, 105 123, 142

+ _+

+/-

58, 85 58

recCrecB- recC-

recAlexApolAtrpt r p - A 2 C ~c recA-

K12,Xclts857 d Xd

B. subtilis 168

recexc rec exc

_ -_

+/_ -+/_

H17 M45

rec + rec-

+ +

+ +

a b c a

h h

u v r B - a n d d e e p r o u g h , except T A 1 9 7 8 , w h i c h is uvrB +. F o r w a r d m u t a t i o n to a r a b i n o s e resistance. F o r w a r d m u t a t i o n to L-azetidine-2-carboxylic acid resistance. P r o p h a g e inductest. W i t h ( + ) or w i t h o u t ( - ) liver $9. t L i v e r $9 or isolated h e p a t o c y t e s . s Positive effect + , w e e k positive ( + ) , n o effect - , b o t h positive a n d n e g a t i v e results + / - . h Positive in p l a t e i n c o r p o r a t i o n assay, n e g a t i v e in spot test.

37 37 37 91 91

191

conditions which give no or only minimal activity when either preparation is used separately [95]. These findings indicate that the conversion of 2NF to 2AF by anaerobic bacteria is probably not the full explanation for the reduction of mutants observed by Goldman and his associates [26] in germ-free rats colonised with Salmonella and B. vulgatus. One possibility might be that the less polar 2AF is readily absorbed through the intestinal lumen and thereby not available for Salmonella. 6. 3. Genotoxic effects in vitro There is a large body of evidence supporting the concept that a major part of known rodent carcinogens are also mutagens. This is also true for 2NF, which induces a number of malignant tumours in rats [103,109] and also causes mutations in most established in vitro assays (Tables 8 and 9). It may, however, be questionable whether the genotoxic effects in vitro are caused by the same 2NF metabolites as the ones being active in vivo. Due to its direct-acting mutagen activity (i.e., active without an exogenous metabolising system) in some Salmonella strains, 2NF has been sug-

gested as a positive control for strain TA1538 and the corresponding plasmid-containing strain TA98 in the Ames assay without a metabolising system [34]. Thus, there are a large number of studies in which 2NF only has been used as a positive control, and those references have not been included in this review. However, it is worth mentioning that there is a marked inter-laboratory variation in the magnitude of mutagenic response to this compound. Although less pronounced, intra-laboratory variations also occur, the variation being larger with TA1538 than with TA98 [29]. Possible reasons for these effects have been discussed by Rosenkranz and Mermelstein [146]. In their review on nitroarenes [146], Rosenkranz and Mermelstein have also discussed in great detail the in vitro genotoxic effects of 2NF, especially in Salmonella. We therefore only briefly summarise the mutagenicity observed with bacteria, and refer the reader to the review above for details. Bacteria Using various Salmonella tester strains in the plate incorporation assay, it has been possible to identify the type of mutagenic damage caused by 2NF. The observation of significant mutagenic

TABLE 9 GENOTOXIC

EFFECTS OF 2-NITROFLUORENE

IN MAMMALIAN

CELLS

Test o r g a n i s m

Assay

Metabolic system a

Genotoxic effect e

References

Mouse lymphoma Chinese hamster Don Chinese hamster ovary C h i n e s e h a m s t e r V79

L5178Y T K + / SSB 8 - a z a g u a n i n e resistance SCE 6 - t h i o g u a n i n e resistance

+ + + b

+ + + + (+)

5, 119, 189 44 48 114 185 185

Hamster embryo

Cell t r a n s f o r m a t i o n

Hamster BHK-21 Human HeLa Rat hepatocytes

Cell t r a n s f o r m a t i o n D N A synthesis i n h i b i t i o n UDS

+ c + + + d

+ + + _

135 135 173 130 140, 141

SSB, single-strand breaks, SCE, s i s t e r - c h r o m a t i d exchange, U D S , u n s c h e d u l e d D N A - s y n t h e s i s . a W i t h ( + ) a n d w i t h o u t ( - ) rat liver $9 in the assay. b I s o l a t e d rat h e p a t o c y t e s i n c l u d e d in the assay i n s t e a d of $9. c I s o l a t e d h a m s t e r h e p a t o c y t e s i n c l u d e d in the assay i n s t e a d of $9. a T h e isolated h e p a t o c y t e s act as b o t h m e t a b o l i s i n g s y s t e m a n d genetic target. Positive effect + , w e a k p o s i t i v e effect ( + ) , n o effect - .

192

activity for the Salmonella strains TA1538 and TA98 is indicative not only of frame-shift mutations due to intercalation in DNA, but apparently combined with the formation of covalent adducts with DNA base pairs. Further support for the formation of a covalent adduct between D N A and the 'activated' fluorene is found in the substantial decrease in activity with strain TA1978, the uvrB + analogue of TA1538 [7,89,90,99,140,145,161,166, 174]. Some activity has also been observed in strain TA1537, which is an indicator strain for frame-shift mutagens causing mutations as a result of intercalation without adduct formation [69]. 2NF causes no or only minimal mutagenic activity in TA1535, whereas TA100 shows some mutagenic response, which presumably reflects a loss of mutagenic specificity as a result of the introduction of the pKM101 plasmid into strain TA1535 [90]. Reduction of the nitro group to the hydroxylamines, followed by esterification to form an electrophile, which can react with the guanine moiety of DNA, has been suggested as the basis of the mutagenic activity of nitrofluorenes [99]. The metabolism of 2NF is further discussed in section 8.3. The ability to induce forward mutations in Salmonella has also been studied with 2NF, which gives a positive mutagenic response in the arabinose (Ara) forward assay [51,149]. Forward mutation to 8-azaguanine resistance is also induced by 2NF, the sensitive strains being TA1535, TA1537 and TA1538. However, strains TA98 and TA100 were negative in the forward-mutation assay, possibly due to the presence of the R factor [21,27]. 2NF has been tested for its ability to preferentially inhibit the growth of D N A repair-deficient strains of E. coli and Bacillus subtilis. The activity is, however, dependent upon the type of repair defect in the D N A repair pathway, and both positive and negative results have been reported (Table 8). In total, 2 recombination-deficient strains have given positive results when exposed to 2NF [61,84,85,92], and 4 have given negative results [176]. Whereas 2NF was inactive towards 2 DNA-polymerase-deficient strains in 3 different studies [42,84,163], it was active in 3 other strains when tested in a modified liquid suspension [92,148].

2NF is positive and 2AAF negative in the E. coli rec A assay [84,85] and rec B assay [61] but both are devoid of activity in the E. coli pol A assay [84] and the E. coli K12 prophage )~ inductest [58,85]. However, in the E. coli K12 )~clts

857 prophage test, 2NF was positive both with and without activation [58]. 2AF, on the other hand, was active in both W P 2 / W P 1 0 0 rec assay and W P 2 / W P 6 7 pol assay [84], but negative in the prophage )~ inductest [85]. 2NF induces a weak SOS response when incubated with rat fiver $9. 2NF without a metabolising system was not tested [123,142]. 2NF caused no induction of tryptophan reversion in E. coli WP2 [105], whereas mutations were induced in CM891 [104,105]. Forward mutation to L-azetidine-2-carboxylic acid ( A 2 C r) ( E. coli WP2 and CM891) was also induced by 2NF [104,105]. A number of repair-deficient strains of B. subtilis have been tested with 2NF. Increased lethal damage was produced in 2 strains of B. subtilis (rec E4 and fh 2006-7), whereas 4 strains were not affected [37]. In a microsuspension adaptation of the B. subtilis rec assay, a DNA-damaging effect of 2NF was detected in the presence of metabolic activation with rat liver $9 [91]. Fungus and yeast

2NF (50-2000 /zg/ml) and other nitro compounds tested are negative in the 8-azaguanine forward mutation of the fungus A. nidulans, probably due to the lack of active nitroreductases [20]. 2NF is also negative when tested in stationary cultures of the yeast Saecharomyces cerevisiae D7, which is less sensitive than Salmonella in detecting frame-shift mutagens [10]. However, 2NF and 2NOF reverted both frame-shift and base-pair substitution mutants of S. cerevisiae in a semiquantitative spot-test employed for the reversion analysis [35]. Increase in mitotic recombination in S. cerevisiae D3 was studied with and without $9. 2NF was recombinogenic, like 2AF, N - O H - A A F and N-acetoxy-AAF, whereas 2AAF, 1-OH-, 3OH-, 5-OH-, and 7-OH-AAF and 4AAF were negative [160,161]. Mammalian cells

Mouse lymphoma cells have been employed to study induction of forward mutations and single-

193 strand breaks in DNA, and in both assays a positive response was obtained without the addition of a metabolising system. 2NF is positive in the mouse lymphoma L5178Y thymidine kinase (TK) assay without metabolising system, giving a 2-3-fold increase in mutation frequency at a dose range of 250-850 /~g 2 N F / m l incubation system [5,119,189]. In one of the studies [5] fluorene and 2AF were negative, whereas 2AAF and Nacetoxy-2AAF were positive in the T K assay without $9. In one of the other studies [189], 2AAF required metabolic activation in order to give a positive result. 2NF causes a doubling of the single-strand D N A proportion and a toxicity of less than 5% in mouse lymphoma cells [44], measured with the alkaline unwinding technique described by Ahnstri3m [1]. 2NF caused a significant increase in reversion frequency over the control level in one 8azaguanine-resistant Chinese hamster Don (CHD) clone (ICR-172) [48]. 2NF also induced SCEs (12 SCEs/cell at 10 /~M 2NF vs. 8 SCEs/cell in controls) in Chinese hamster ovary (CHO) cells without metabolic activation. The addition of Aroclor 1254-induced rat liver $9 mix trebled the number of SCEs/cell [114], which is contrary to the results reported in Salmonella by Wang et al. [192], who found that $9 mix led to a decrease in the mutagenic activity of 2NF. This difference in results could either be due to differences in the $9 fraction used (different enzyme contents), or to the fact that bacterial metabolism differs from that of mammalian cells [114]. The effect of metabolic conversion of 2NF is further discussed in section 8.3. The mutagenic activity of 2NF has been investigated with Chinese hamster V79 cells (6thioguanine resistance). Only marginal effects were produced in the presence of isolated hepatocytes and no effect with a subcellular liver preparation or in the absence of metabolising system. 2AF and 2AAF were only mutagenic at high concentrations and in the presence of hepatocytes [185]. Isolated rat hepatocytes have been used to study the induction of D N A repair measured autoradiographically as UDS. 2NF does not induce UDS in isolated rat hepatocytes in vitro [140,141], although in vivo treatment of rats with 2NF (10-50 m g / k g body weight orally) gives rise to UDS

measured in hepatocytes isolated from the exposed liver [15,16 and section 6.1]. The induction of D N A lesions due to exposure of HeLa cells to mutagens results in an inhibition of the overall rate of D N A synthesis even after the removal of the genotoxic agent. When 2NF was tested in this assay, it inhibited the HeLa D N A synthesis at a concentration 3 × 10 - 3 M. However, 2NF was only tested in the presence of Aroclor-induced rat liver $9 [130]. Morphologic cell transformation is generally associated with oncogenic properties. Thus, 2NF was studied in the hamster cell clonal transformation assay. Transformed colonies were induced by 2NF (20 /~g N F / m l ) in the presence of isolated hamster hepatocytes, whereas no transformed hamster embryo cells were obtained in the absence of metabolic activation system [135]. Induction of cell transformation has also been demonstrated with hamster BHK-21 cells in the presence of $9 [173]. 6.4. DNA adducts The principal D N A adduct resulting from exposure of TA1538 to the potential 2NF metabolite, 2-OH-AF, is N-(deoxyguanosin-8-yl)-2aminofluorene [18], and this adduct can be easily accommodated in the major groove of the D N A helix [186]. Furthermore, it has been shown that the mutagenicity in Salmonella of the aryl hydroxylamine intermediate of 2NF is directly proportional to the extent of arylation at the C8 position of the DNA-guanine moiety [86]. Using Salmonella strains TA98 and TA97, Rosenkranz and coworkers [143] have demonstrated that C8 is the preferred site of adduct formation. This adduct is biologically significant with respect to carcinogenesis and mutagenesis [17,18,59]. 2NF also induced mutagenicity in the Salmonella strain TA96, which contains a stretch of 5 adenines at the mutational site [79], indicating that 2NF reacts not only with guanine, but also to a minor extent with adenine [78,86]. The mutagenic activity of 2NF is not decreased after 5 h incubation with pure DNA, and neither is the mutagenicity of diesel particle extract. This has been taken as an indication that 2NF and the direct-acting bacterial mutagens in the diesel par-

194 ticle extract do not bind covalently to purified D N A without metabolism [132].

6.5. Summary of genotoxicity The genotoxic activity of 2 N F in vivo seems to be dependent on the route of administration. Out of 4 studies using the oral route, 3 have given. positive results, i.e., liver UDS, bone marrow SCE and urinary and faecal mutagenicity. Three studies using intraperitoneal injections (bone marrow SCE and M N and sperm-head abnormalities) were all negative. However, contrary to these data, 2 studies with host-mediated assay (TA1538) have given the opposite results. Thus, after oral administration of 2NF, no TA1538 mutants were recovered, while intramuscular injection gave a positive result. When germ-free rats have been used to eliminate intestinal microbiological biotransformation, the mutagenicity excreted in urine and faeces is markedly higher than that obtained with conventional rat strains. This is in agreement with the results observed with germ-free rats associated with TA1538 (as mutagenic indicator) and some bacteria representative of normal gut flora. 2 N F treatment gave a lower revertant response compared to germ-free rats associated only with TA1538. In vitro studies with mammalian cells show predominantly positive results (7 out of 8). In some cases, exogenous metabolic activation has been necessary, i.e., 6-thioguanine resistance in V79 cells and hamster embryo cell transformation were only induced in the presence of isolated hepatocytes, while the addition of liver $9 was sufficient for D N A synthesis inhibition in H e L a cells. In 4 assays, 8-azaguanine resistance in Chinese hamster D o n cells, SCE in Chinese hamster ovary cells, and SSB and L5178Y T K in mouse l y m p h o m a cells, a positive response was induced without exogenous metabolic activation. N o induction of U D S was detected' in isolated hepatocytes exposed in vitro to 2NF, despite the fact that addition of hepatocytes was necessary for the induction of mutations in V79 cells and cell transformation in hamster embryo cells. Also in bacteria (S. typhimurium, E. coli, B. subtilis) the positive results were in the majority

(26 positive and 15 negative), the negative ones being primarily due to less sensitive genetic strains. The positive results in the bacterial assays were not dependent on the addition of a metabolising system, except when nitroreductase-deficient Salmonella strains were employed. In this case liver $9 or isolated hepatocytes improved the effect. The fungus A. nidulans did not respond to 2 N F treatment, whereas forward mutations and mitotic recombination were induced in the yeast S. cerevisiae.

7. Induction of preneoplastic lesions 2 N F has been assayed in an in vivo model with pre-neoplastic lesions in the rat as end-point. When 2 N F was given orally, a dose-response curve was obtained for initiating activity measured as number of loci in the liver. This activity was significant relative to controls. When 2 N F was given orally as a promoter, and the end-point was the relative number of foci, a significant dose-response curve was also obtained. However, 2 N F was a weak promoter compared to 2AAF (L. MiSller et al., unpublished).

8. Biotransformation of 2-nitrofluorene 8.1. Mammalian metabolism in vivo Conventional rats given a single dose of 2 N F (25 m g / k g body weight) by oral gavage excrete 90-96% of the dose within 4 days. Approximately 2 / 3 of the excreted metabolites appear in the urine and 1 / 3 in the faeces [112]. By comparison, 1-nitropyrene administered orally is mainly excreted in the faeces [12]. Using H P L C and L C / M S , a number of urinary 2 N F metabolites have been characterised. A major part of the free, i.e., unconjugated, metabolites consists of hydroxylated 2AAFs (7-OH- and 5-OH-AAF, and to a lesser extent 9-OH-, 8-OH-, 3-OH-, 1-OH- and N - O H AAF), all of which have also been identified as metabolites after 2AAF administration. However, 2AAF per se has not been detected in the urine after oral administration of 2NF, and neither is it known to be excreted in the urine after 2AAF administration [193[. Apart from hydroxylated

195 2AAFs, hydroxylated 2NFs have also been identified in urine. The position of the hydroxyl group in the hydroxylated N F s is not known, except that 9 - O H - N F can be excluded. The H P L C profiles of methanol extracts of faeces indicate the presence of the same metabolites as in urine, i.e., hydroxylated 2AAFs and 2NFs. In addition, minor amounts of unmetabolised 2 N F were detected in faeces, whereas no 2 N F was excreted in the urine. Part of the hydroxylated 2 N F metabolites was excreted as glucuronides in the urine, while the 2 N F metabolites in the faeces were mainly presented in an unconjugated form. It is well established that 2AAF metabolites are excreted in the bile mainly as glucuronides [193]. It seems, there-

fore, reasonable to assume that O H - N F s are also excreted as conjugates, which are then split by the gut microflora. Support for this assumption is found in section 8.2. The major 'direct-acting' mutagenic activity in urine, as discussed above (section 6.1), was associated with the unconjugated 2 N F metabolites. A large part of the mutagenicity was probably accounted for by N - O H - A A F and O H - N F . In faeces the main part of direct-acting mutagenicity was most likely due to O H - N F s and unmetabolised N F [112]. In rats treated with fl-naphthoflavone (induces cytochrome P-450c and d) prior to the 2 N F treatment, the metabolic pattern shifted towards excre-

~ wf#,~12

.A

/

= o

N

l

/ / •

•

~.,,_..,

Excretion

~

",

\ \\

o.

/ \

/ + onjug.

.... -

.~o. -L(.

With/without

2 m

,,>

",

I

. . . . .

/

- -

""

Liver/Lung

.

-

\

~.:~..

-

"-

xcretion

\ --~'-./--~.o=1o.

/

~ ( ~ ~

\

/

~.~ l

Excretion

coniuqalhon

"

Fig. 3. Tentative scheme for the metabolism of 2-nitrofluorene (2NF) in conventional rats (with and without fl-naphthoflavone (4f l N F ) induction), and germ-free rats, as well as isolated, perfused rat liver and lung (Liver/Lung). In vivo, 2 N F was administered by oral gavage. In isolated liver perfusion, 2 N F was administered via the portal vein, and in isolated lung perfusion either intratracheally or intravascularly. In vivo, the metabolites are excreted in the urine and the faeces. In the isolated liver perfusion, the hydroxylated 2 N F s are excreted in the bile as conjugates. In the isolated lung perfusion, the hydroxylated 2NFs are released to the perfusate as free metabolites. A - A A F indicates N-acetoxy-2-acetylaminofluorene, 1-OH-AAF and 3 - O H - A A F indicate 1- and 3-hydroxy-2acetylaminofluorene, respectively.

196 tion of a larger proportion of hydroxylated 2NFs compared to uninduced rats, and simultaneously the mutagenicity of urine and faeces increased [110,112]. The excretion pattern in germ-free rats differs considerably from the one described above for conventional rats. After a single oral dose of 2NF, no hydroxylated 2AAFs are detected either in the. urine or in the faeces. Instead, several mono- and di-hydroxylated 2NFs were identified [111], including 9-OH-NF, a metaborite that is also produced by the isolated, perfused liver and lung [113, and section 8.2]. Only trace amounts of unmetaborised 2NF were detected in the urine and faeces of germ-free rats. As mentioned above, the excretion of hydroxylated 2NFs was associated with a higher direct-acting mutagenicity compared to the mutagenicity excreted by conventional rats. On the basis of the urinary and faecal metaborites discussed, a tentative scheme is presented in Fig. 3, which summarises the various pathways for 2NF metabolism in conventional and germ-free rats. Five daily i.p. injections of 2NF or 2AAF (224 /~m o l e / k g body weight) to male SpD rats increased the amount of cytochrome P-450c in the river microsomes about 15-19 times, whereas the same treatment with 2AF induced a 60-fold increase in P-450c. A significant increase in P-450d (up to 3.9 times) could be seen in liver microsomes from rats treated with 2NF, 2AF or 2AAF, and the same compounds increased the microsomal content of epoxide hydrolase [206]. 8.2. Metabolism in isolated organs

The isolated perfused lung metabolises 2NF (50 /~g) to hydroxylated 2NFs, mainly 9-OH-NF and X - O H - N F (the position of the OH-group not known), whether administered to the perfusate or intratracheally. By the latter route, 2NF is rapidly absorbed into the circulating perfusate [113], with nearly 80% of the given dose appearing in the perfusate within minutes of administration. This implies that after exposure to 2NF in the air, the inhaled compound may r a p i d l y be distributed within the body. When 2NF (200 /tg) is administered to the isolated perfused liver, 70% of the dose is taken up

by the liver within 10 min, and the first metaborites in the bile are detected at this time point. The biriary 2NF metaborites excreted are hydroxylated and conjugated (glucuronides), which supports the assumption that the metabolites are delivered to the gut as conjugates, which are subsequently sprit. Treatment of the bile with fl-glucuronidase gave rise to direct-acting mutagenic activity in the Ames assay with TA98, and the metabolites identified were the same as those in the perfusate of the isolated lung, i.e., 9-OH-NF and X-OH-NF [113]. 8.3. Metabolism in vitro Bacteria

The major part of the 2NF-induced mutagenic activity observed in bacteria is dependent on reduction of the nitro group. This reaction takes place through nitroreductases present in most bacteria. There is evidence for the presence of at least 2 different sets of nitroreductases, one that activates nitropyrenes, 4-nitroquinoline-l-oxide and nitroacridine and also the 'classical' nitroreductase which activates nitrated fluorenes and fluorenones as well as nitrofuranes and nitronaphthalenes [96,144,145,148]. The hypothesis that reduction of the nitro group is important for the expression of the mutagenic activity in bacteria is supported by studies with nitroreductase-deficient Salmonella strains, i.e., TA1538-FR1, TA98NR and TA100FR. These strains do not respond, or show a substantially reduced response, to the mutagenic action of a number of nitro-containing substances, including 2NF and 2-nitronaphthalene [8,28,94,99,145,148]. Furthermore, Wang and coworkers [71,190] have shown that 2-OH-AF is more active (approximately 8 times) than the corresponding nitro compound in strain TA98 while its mutagenicity is unchanged in strain TA98NR. It has also been demonstrated that chemical reduction of 2NF to the corresponding hydroxylamine by treatment with zinc dust and ammonium chloride results in a significant mutagenic activity in the nitroreductase-deficient strain TA98NR [43,49,99,167]. These results indicate that 2-OH-AF could be an intermediate metabolite in the bacterial reduction of 2NF. Studies with T A 9 8 / 1 , 8 - D N P 6 indicate, furthermore, that esterification to hydroxamic acid

197 esters might be necessary [93]. On the other hand, the unchanged mutagenic activity in strains TA1537 and TA1537NR indicates that reduction of the nitro function is not required for the expression of intercalation [145].

Liver fractions A rat liver microsomal preparation has been shown to have the ability to convert 2 N F to metabolites mutagenic in Salmonella strains deficient in the 'classical' nitroreductase [148]. However, in another study it was demonstrated that the addition of Aroclor-induced rat liver $9 to nitroreductase-proficient Salmonella reduced the mutagenicity of 2 N F (6.25 /~g/plate) to about 25%, and a similar effect was seen with diesel exhaust samples. The antimutagenic activity was shown to be non-enzymatic, 90% of the effect being accounted for by the amount of albumin present [191]. When 2 N F is incubated with rat fiver microsomes and an N A D P H - g e n e r a t i n g system under anaerobic conditions, a nitroradical anion is formed as detected with electron-spin resonance spectroscopy (esr). The esr signal disappears when the system is purged with air. By addition of a spin trap, variable amounts of superoxide and hydroxyl free radicals were detected during the aerobic interaction of 2 N F with microsomes and N A D P H [167]. Hamster fiver $9 does not increase the directacting mutagenic activity (Salmonella TA98) of 2 N F (70 r e v / n m o l e without $9 and 50 r e v / n m o l e with $9) or 7 - O H - 2 N F (approximately 50 r e v / n m o l e , with or without $9), whereas the addition of $9 was essential to obtain mutagenicity with 2AF (16 r e v / n m o l e ) and 2AAF (4.5 rev/nmole). While the mutagenic activity of 2AF was maintained in the nitroreductase-deficient strain TA98NR, it was markedly reduced with 2AAF and 2NF. With 7-OH-2NF, however, the reduction was only approximately 50% in the presence of $9, but more than 10-fold lower in the absence of $9 [187]. PCB-induced and 3-methylcholanthrene-induced rat liver homogenates decompose 2 N F at approximately the same rate (90-95% at 2 h), whereas phenobarbital-induced rat liver homogenate was much less effective ( < 60% decomposed at 2 h).

Lung fractions Incubations of 2 N F with subcellular fractions (microsomes, cytosol, submitochondrial particles) from rat lung demonstrated that the rate of superoxide generation was increased in a dose-dependent manner in the presence of microsomes [157]. The data indicate that 2 N F has the potential to generate superoxide in rat lungs.

Prostaglandin H synthetase The prostaglandin H synthetase (PHS) activity of microsomes from ram seminal vesicle has been used as an alternative to the rat liver postmitochondrial ($9) fraction in the Ames assay, and among the most efficient substrates are aromatic amines. Thus the metabolism of 2AF has been studied in great detail and the products isolated were 2,2'-azobisfluorene, 2-aminodifluorenylamine and 2NF. When activated by PHS, 2AF produced the same number of revertants in Salmonella TA98 and TA98NR. This indicates that the observed mutagenic effect is not primarily due to the effect of nitroreductases as discussed above, but possibly due to some other mechanism giving rise to 2AF free radical species [22].

8.4. Summary of biotransformation In vivo studies in conventional rats have indicated that a major metabolic route for orally administered 2NF is via reduction to 2AF (by nitroreductases in the gut microflora), which is then subsequently converted in the liver to C- and N-hydroxylated 2AAFs. These are excreted, mainly as conjugates, in the urine and faeces. A minor metabolic route involves direct absorption of 2NF, which is then hydroxylated and conjugated in the liver and excreted in urine and faeces. Induction with fl-naphthoflavone causes a metabolic shift towards a larger proportion of hydroxylated 2NFs. In germ-free rats, mono- and di-hydroxylated 2NFs have been identified in urine and faeces after 2 N F ingestion, whereas no 2AAF metabolites have been detected. When the isolated perfused liver is exposed to 2NF, a similar metabolic pattern to the one described for germ-free rats is observed. However, as the metabolites are collected directly from the bile, they all appear as conjugates. The isolated

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perfused lung metabolises 2 N F to the same hydroxylated 2NFs as the isolated perfused liver, although less efficiently, and no O H - N F conjugates are formed. In bacteria the biological activity of 2 N F seems to depend on the conversion to arylhydroxylamines, which are either direct-acting or become so following esterification to electrophiles. Mammalian cells are also susceptible to the mutagenic and genotoxic actions of 2NF. However, this may occur by a variety of mechanisms including nitro reduction, ring oxidation or a combination of the two. In subcellular liver fractions there seems to exist a reversibility between the production of 2AAF metabolites and 2AF on one hand, and 2 N O F and 2 N F on the other, depending on the incubation conditions used. 9. Modulators of 2-nitrofluorene mutagenicity in vitro It is well established that chemical carcinogens can be influenced by compounds which promote or inhibit carcinogenesis. The Ames assay has provided a simple means to screen potential modulators of mutagens, and some biological substances have been investigated in connection with 2NF, for example hemin and its metabolites biliverdin and bilirubin [9], the cysteine precursor N-acetylcysteine (NAC) [197] and 2 secondary bile acids [198]. The mutagenic activity towards Salmonella TA98 and TA100 of 2 N F as well as several other carcinogens (i.e., b e n z o [ a ] p y r e n e , 3-methylcholanthrene, 7,10-dimethylbenz[ a ]anthracene, chrysene, 2-AAF, aflatoxin B1) is markedly reduced by hemin. Generally, 1 - 2 equivalents of heroin to the mutagen caused 50% inhibition. Biliverdin and bilirubin, on the other hand, were only effective as inhibitors of benzo[a]pyrene mutagenicity [9]. Wilpart et al. [197] have studied the effect of N A C on 2 N F mutagenicity. In the Ames test, N A C inhibited the mutagenic activity of 2 N F towards both TA98 and TA1538. N A C also inhibited 2-aminoanthracene. N A C seems to act on the activated intermediates rather than on metabolising enzymes [197]. The same group also studied the co-mutagenic activity of the 2 sec-

ondary bile acids, lithocholic acid and deoxycholic acid, in the Ames assay (TA1538) with 2 N F and 2AAF. While lithocholic acid inhibited the mutagenic activity of 2 N F (55%), it was not modified by deoxycholic acid. Lithochofic acid and deoxycholic acid are interesting in this connection as they constitute the 2 major bile acids found in faeces [198]. Other inhibitors of 2 N F mutagenicity have been identified. Methylene chloride extracts of cooked pork contain material which inhibits or suppresses the mutagenic activity of 2 N F as well as M N N G and 2-aminoanthracene [51]. Two chalcones caused a 10-30% inhibition of 2 N F mutagenicity (TA98, TA100), whereas one chalcone had no effect [182]. Several known suppressors of chemically induced mutations have been shown to be inactive towards 2 N F mutagenicity. Complex mixtures of aromatic compounds isolated from a coal-derived oil, which suppress the mutagenic activity of 2AF and 2AAF, as well as benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, (Salmonella TA98 + Aroclor-induced rat liver $9), have no effect on the mutagenic activity of 2 N F and benzo[a]pyrene-diol-epoxide [54]. A bio-antimutagen isolated from Japanese green tea reduces high spontaneous mutations due to altered DNA-polymerase III in a mutator strain of B. subtilis, but had no effect on the mutagenicity of several mutagens tested including 2 N F [67]. Extracts of wheat sprouts exhibit antagonist activity towards known carcinogens (2AAF, B(a)P, 3MC and aflatoxin B1) that require metabolic activation, whereas no inhibition was observed for 'direct-acting' mutagens like 2NF, EMS and M N N G . The extracts were most active in altering the primary metabolic pathway of 2AAF and less active in inhibiting its derivative N - O H - A A F and 2AF [77]. We have only found one study in which an enhancement of 2 N F mutagenicity has been demonstrated in vitro. The antischistosomal drug, praziquantel, enhanced the mutagenic activity of 2NF, M N N G , 9-aminoacridine and B(a)P in a dose-related fashion, when measured as 8-azaguanine resistance in TA100 [41]. Other compounds that enhance mutagenic activity have been investigated in several studies. However, none of the substances showed any ef-

199 fect on the mutagenicity of 2NF. A desmutagenic factor for Trp-P-2 and Trp-P-1 which was identified in extracts from cabbage (Brassica oleracea) had an enhancing effect on the mutagenicity of 1,2-diamino-4-nitrobenzene, a direct-acting mutagen in Salmonella TA98 and TA100. However, neither 2NF nor any of the other chemically related amino- and nitro-aromatics investigated were affected [63]. Norharman enhances the mutagenicity of 2AAF and N - O H - A A F in the presence of a metabolising system, and N-acetoxy-AAF without a metabolising system, in the Salmonella test system, but has no effect on the mutagenicity of N-OH-AF, 2NF or benzo[a]pyrene-4,5-oxide in the absence of $9 [184]. Pyrenequinones, which enhance the mutagenicity of 2AF and 2AAF in the presence of $9, have no effect on the mutagenic activity of 2NF and N-OH-AF in the absence of $9 [125]. Finally, no enhancing effect on the mutagenicity of 2NF, N - O H - A F and Nacetoxy-AAF towards Salmonella TA98 was observed in the presence of sucrose pyrolysate, although the mutagenic activity of 2AAF, 2AF, AA and Trp-P was enhanced by $9 [124]. 10. Genotoxic effects of nitrofluorenones and nitrofluorenes other than 2NF

Among the nitrofluorenes, 2NF has the best documented prevalence and biological effects. However, other nitrofluorenes have been identified in the environment, such as dinitrofluorene, 2NFone, 2,7NFone and 2,4,7NFone. These compounds have mainly been studied for their mutagenic activity in bacteria. The mutagenic activity of nitroarenes in the Salmonella strains TA1538 and TA98 increases with the number of nitro groups: 2NF < 2,7NF < 2,7NFone < 2,4,7NFone [94,73]. However, with a fourth nitro group (2,4,5,7NFone), the mutagenic activity is lower than with 3 nitro groups. Excessive nitration of other PAHs gave similar results, for example tetranitronaphthalene and tetranitropyrene [145]. Oxidation of carbon 9 in the fluorene molecule, as in 2,7NFone, results in increased mutagenicity in strains TA1538 and TA98 but a slight decrease in TA1537 as compared to 2,7NF [94,73]. Some fluorene compounds show a modest yet finite activity in strain TA1977 (the uvrB +

analog of TA1537), ranging from 0.06% for 2,7-NF to 1.3% for 2,4,7NFone, thus indicating some intercalative activity [7,145,166]. The mutagenic activity of 2,7NF (346 r e v / nmole in TA98) has been shown to be comparable to that of benzo[a]pyrene-7,8-diol-9,10-epoxide (376 r e v / n m o l e in TA98) [76], while the mutagenic activities of 2,4,7NFone and its malononitrile derivative (approx. 2200 r e v / n m o l e with TA98) approach that of the structurally related Tr-P-2 (3-amino-l-methyl-5H-pyrido[4,3-b ]indole), with 2760 r e v / n m o l e in TA98 in the presence of $9 [1451. 10.1. Di-nitrofluorenes The mutagenic potency of 2,7-dinitrofluorene (2,7NF) in Salmonella TA98 (2300 rev/nmole) has been investigated with different substitutions at the C-2 position, including 2NF (90 rev/nmole). Functional groups at C-2 having a free pair of electrons ( - N H 2 and - O H ) are less mutagenic than 2NF. Acetylation of the amino group at C-2, which decreases the availability of electrons on the nitrogen, increased the mutagenic response to 450 rev/nmole. 1-(N-acetylamino)-7-NF is less mutagenic than 2NF, indicating that conjugation is a factor determining mutagenicity. The nitro groups on 2,5NF also form a conjugated system whose activity (1800 rev/nmole) indicates that deactivation of one of the aromatic rings may be essential to increased genotoxic potency [187,188]. When 2,7NF was incubated with TA98, the addition of rat liver $9 caused a slight increase in the mutagenic activity compared to that without $9. With NF, on the other hand, the mutagenic activity was marginally reduced when $9 was added [74]. When tested in the mouse lymphoma L5178Y T K mutation assay, 2,7NF caused a rise in mutant frequency with increasing concentrations [4]. 10.2. 2-Nitro-9-fluorenone One of the most abundant fluorenes in diesel exhaust particulate extracts (DEPE) is 9-fluorenone and its C1-C4 alkyl homologues. Thus, nitrated 9-fluorenone might occur in DEPE. 2NFone has been synthesised and its mutagenicity was compared to that of 2NF and 1-nitropyrene. It was

200 shown that 2NFone was more mutagenic than 2 N F by a factor of 4 in TA98 and TA98NR, and exhibited about 1 / 5 to 1 / 1 0 of the mutagenicity of 1-nitropyrene [14]. By analogy with the mutagenic potency of dinitrated and mononitrated fluorenes discussed above, 2,7-dinitro-9-fluorenone is more mutagenic to TA98 than 3-nitro-9-fluorenone [66]. 10.3. Trinitrofluorenone 2,4,7NFone is a potent mutagen in bacteria [140,141]. It is primarily a frame-shift mutagen active in TA1538, TA98 and TA1537, but it also induces base-pair mutations in TA100. Metabolic activation is not necessary [23,165]. 2,4,7NFone induces a dose-dependent response in the Ara-resistant forward-mutation assay (Salmonella SV50) [165]. It is also mutagenic in the L5178Y T K mouse lymphoma assay, and causes a significant increase in SCE in C H O cells, both with and without $9 [23,165], as well as SCE and chromosome aberrations in h u m a n peripheral lymphocytes [183]. N o significant genotoxic effects (gene conversion or mitotic crossing-over) were observed in the yeast S. cereuisiae D7, at the 2,4,7NFone doses that could be tested without toxic effects [165]. 2,4,7NFone produced a highly significant mutagenic response in E. coli strain WP2 uvrA-. Fluorenone decreases the aryl hydrocarbon hydroxylase activity, and both fluorenone and 2,4,7NFone decrease epoxide hydrolase activity, which might suggest possible toxicity of these compounds during long-term exposure [57]. 2,4,7NFone induces U D S in rat hepatocytes in vitro [141] and is a powerful carcinogen in female rats, giving rise to an increase in the incidence of m a m m a r y tumours in rats after a single dose administered orally [60]. Intragastric administration of 2,4,7NFone to rats led to the excretion of mutagenicity in the urine during the first 24 h, whereas subcutaneous exposure caused prolonged urinary mutagenicity. In both cases, however, only a minor amount of the dose was excreted via urine in a form which retained mutagenic properties [32]. Analysis of h u m a n body fluids for the presence of mutagenic compounds is used in the detection of exposure to genotoxic agents, although a nega-

tive result cannot be regarded as proof of no exposure. Urinary mutagenicity in workers occupationally exposed to 2,4,7NFone did not show any relationship to the presumed exposure [32]. 10. 4. 2-Nitrosofluorene Incubation of N - O H - A A F with rat liver microsomes or alternatively with horseradish peroxidase and H202 has been shown to give rise to 2NOF, and in the latter case also N-acetoxy-AAF [13]. Furthermore, the kinetics of 2AF photo-oxidation has been studied with H P L C and the Salmonella mutagenicity bioassay using the tester strains TA98, TA1538 and TA98NR. The results indicate that photo-induced mutagenicity of UVA-irradiated 2AF can to some extent be attributed to the rapid formation of 2NOF. However, the majority of mutagenic activity was due to the formation of 2 N F [127,128,171,186]. Other studies have demonstrated the rapid conversion of 2NOF. Thus, conversion of 2 N O F to 2AF has been suggested to take place as a result of enzyme-mediated reduction by rat liver homogenate, the observed mutagenic effect being 400-500 m/~mole/40 mg rat liver/20 min [83]. In another study, it has been shown that 2 N O F added to a 150000 x g supernatant from rat liver is rapidly converted to N - O H - A F by a non-enzymatic reaction [169]. 2 N O F has been shown to be a more active frame-shift mutagen than 2 N F [6], and the mutagenicity of 2 N O F is almost the same in TA98 and TA98NR, which suggests either that 2 N O F is a direct-acting mutagen or that it requires further metabolism by a bacterial enzyme other than the 'classical' nitroreductase. The mutagenicity of both 2 N F and 2 N O F is greatly reduced in the nitroreductase-deficient strain, TA98/1.8-DNP6, compared to TA98. The results suggest that the reduction of nitro-containing chemicals to mutagenic hydroxylamines may involve 2 separate enzymes [94]. King and Phillips [70] have studied the formation of adducts between N - O H - A A F , N - O H - A F and 2NOF. The 2 aminofluorene derivatives formed adducts with guanosine, whereas no such adducts were detected with 2NOF. On the other hand, 2 N O F reacts readily with sulfhydryl groups

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[83], and is, like 2AF and 2AAF, a potent inhibitor of amino acid incorporation into proteins [11,100]. When mapping histidine revertants induced by mutation with several compounds in strain TA1538, Isano and Yousno [64] found that revertants induced by 2NOF invariably resulted from the deletion of a - G - C - doublet from the D N A sequence close to the 3052 site. The deletion of this doublet produced a 3 deletion mutant with a correct reading frame. 2NOF increased the frequency of 8-azaguanine resistance in diploid, human, foreskin-derived flbroblasts [65], but it failed to increase the frequency of deletion mutants (gal-chl) in Salmonella [3]. When fed to male Drosophila larvae, 2NOF did not induce a frequency of lethals that was significantly different from the control, and none of the lethals were temperature-sensitive [200,201]. 11. Mutagenicity of ambient air and diesel exhaust The presence of mutagenic activity has been demonstrated in samples from diesel exhaust [31,116,133,134,202,203,205], ambient air [2,45, 158] and airborne particulates [55,68,137,139,192], in coal fly ash [30,52,75,194], and in the emission from kerosene heaters [177]. Sediment from the Suimon river in Japan has also been shown to be mutagenic to Salmonella [151]. The mutagenicity of extracts of airborne particulates is partially associated with the presence of nitro-PAHs, of which nitropyrenes have been identified in some samples. In the air samples from southeast Michigan, Siak and collaborators [158] have detected 1NP, and 1,6- and 1,8-dinitropyrene, but these 3 compounds could only account for 3% of the total airborne mutagenicity. In other studies, indirect evidence for the presence of nitroarenes has been obtained by including the n i t r o r e d u c t a s e - d e f i c i e n t S a l m o n e l l a strain T A 9 8 N R in the test protocol [71,137]. There is evidence that over 87% of the mutagenic activity (TA1538, TA100, TA98) in coal fly ash is associated with the nitro-organic fraction [194]. Mutagenicity assays indicate, furthermore, that 1NP accounts for only 0.03-0.16% of the total activity of coal fly ash [52].

In air samples collected after the ignition of a kerosene heater, 40-80% of the direct-acting mutagenicity was associated with dinitropyrenes and 1NP. However, other nitroarenes, such as 2NF, dinitronaphthalene and 4,4'-dinitrobiphenyl, were also identified in the samples, but their contribution to the mutagenicity of the extracts was low [177]. In an extract of diesel exhaust particulates, more than 50 nitro-PAHs have been tentatively identified. Positive identification of each nitroPAH is difficult due to the small quantities of nitro-PAH relative to co-eluting oxygenated and sulfur-containing PAHs. Among the nitroarenes identified are predominantly nitropyrenes and nitroanthracene [116], but 2NF and dinitrofluorene have also been detected [202]. In their study on the mutagenic activity of diesel particle extract, Pedersen and Siak [134] recovered 1 / 3 of the mutagenicity in fractions containing monosubstituted nitro-PAHs, 1NP being the main component, but not the only mutagen in those fractions. In another study, 1NP, 2,7NF and a diesel extract were investigated in the Salmonella plate incorporation assay (TA98). 1NP and 2,7NF contributed less than 1.5% of the mutagenic activity of the diesel exhaust extract [74]. Organic extracts of the sediment from the Suimon river in Japan have been shown to have a direct-acting mutagenic effect towards Salmonella TA98, whereas the mutagenicity towards the nitroreductase-deficient strain, TA98NR, was the same as the spontaneous mutation level. When the mutagenicity of the organic extracts of the sediment was compared with a mixture of 2NF, 1NP, and 4,4'-dinitrobiphenyl, in the same concentrations as in the sediment, the results were concordant [151]. Lung macrophages recovered from rats exposed to diesel particles have no detectable mutagenic activity even though the diesel particles were recovered [159]. This indicates that the mutagenic compounds attached to the diesel particles are absorbed in the lung tissue. Furthermore, it has recently been demonstrated that rats chronically exposed to diesel exhaust (at soot concentrations of 3.5 and 7.0 m g / m 3 for up to 30 months) by inhalation developed malignant lung tumours. The total percentage of rats with lung tumours at the

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higher exposure level was 12.8%, and at the lower dose 3.6%, both of which were significantly higher than that of controls [88]. In this connection it might be worth noting that, in their cancer bioassays, Morris et al. [109] detected 2 lung tumours among 7 rats exposed to 2NF by painting, and Miller et al. [103] detected 1 rat with lung tumour among 9 male rats exposed to 2NF in the diet. So far, it has not been possible to associate the major part of the mutagenicity in air samples or diesel samples with a particular compound, although nitro-PAHs undoubtedly play an important role. One of the nitro-PAHs that has been identified in ambient air, diesel exhaust and emission from heaters, is 2NF, which has also been shown to induce malignant tumours in rats treated either orally or by skin painting. Furthermore, exposure to diesel exhaust induces lung tumours in rats, and so does 2NF. In view of the data from in vitro as well as in vivo genotoxicity, and the available cancer data with 2NF and diesel exhaust, it seems likely that 2NF is a contributing factor as a potential health hazard to humans.

Acknowledgements The authors wish to express their gratitude to Dr Elizabeth von Halle, EMIC, who provided the lengthy list of references, to Mary-Ann Zetterqvist, B.Sc., for gathering all the references, and to Mrs Maud W~tglund and Inger Bonsdorff for help with the typing. This work has been supported by research grants from the Swedish Environmental Protection Board (B.B. and L.M.), and grants to James Ware (B.B.) and Jan-~tke Gustafsson (L.M.) from the Swedish Cancer Society.

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