Cytogenetic analyses of the in vitro and in vivo responses of murine cells to peroxyacetyl nitrate (PAN)

Genetic Toxicology

ELSEVIER

Mutation Research 341 (1995) 199-206

Cytogenetic analyses of the in vitro and in vivo responses of murine cells to peroxyacetyl nitrate (PAN) A.D. Kligerman a,*, K. Mottus b G.L. Erexson c a Genetic Toxicology Division, Health Effects Research Laboratory, Mail Drop 68, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA b University of North Carolina, Curriculum in Toxicolgy, Chapel Hill, NC 27599, USA c Environmental Health Research and Testing, Inc., P.O. Box 12199, Research Triangle Park, NC 27709, USA Received 26 April 1994; revision received 15 August 1994; accepted 30 August 1994

Abstract Peroxyacetyl nitrate (PAN) is one of a class of common air pollutant formed by the action of sunlight on volatile organic compounds and nitrogen oxides. PAN has been shown to be a bacterial mutagen. To determine if PAN can cause D N A damage in mammalian cells, we exposed murine peripheral blood lymphocytes (PBLs) to various volumes of PAN in vitro and analyzed the cells for chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and D N A damage using the single cell gel (SCG) assay. At in vitro concentrations of PAN that were cytotoxic (inhibited cell division), an increase in D N A damage was noted in the SCG assay. At lower exposure levels that permitted cell division, no increases in SCEs, CAs, or D N A damage were evident. For in vivo studies, male mice were exposed nose-only by inhalation for 1 h to 0, 15, 39 or 78 ppm PAN, and their lung cells removed and cultured for the scoring of SCEs and CAs. In addition, PBLs and lung cells were analyzed by the SCG assay. No dose-related effects were found in any of the assays. These data indicate that PAN does not appear to be a potent clastogen or DNA damaging agent in mammalian ceils in vivo or in vitro.

Keywords: Peroxyacetyl nitrate; Cytogenetic damage; D N A damage; Air pollution; Lung

Introduction * Corresponding author. Tel. 919/541-4254; Fax 919/5414361 or 919/541-0694. Disclaimer: This paper has been reviewed by the Health Effects Research Laboratory of the U.S. Environmental Protection Agency and has been approved for publication. Approval does not signify that the contents reflect the views a n d / o r policy of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Elsevier Science

B.V.

SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 6 3 - 8

M a n y of t h e m a j o r c o m p o n e n t s o f air p o l l u t i o n a r e d e r i v e d f r o m t h e a t m o s p h e r i c o x i d a t i o n of h y d r o c a r b o n s in the p r e s e n c e of n i t r o g e n oxides (NOx). S o m e volatile air p o l l u t a n t s , such as p e r oxyacyl n i t r a t e s [ R C ( O ) O O N O 2 ] , a r e k n o w n to form through photochemical processes rather t h a n c o m b u s t i o n ( K l e i n d i e n s t et al., 1990). Perox-

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yacetyl nitrate (PAN), the simplest m e m b e r of this class, is a common and relatively stable component of polluted atmospheres (Stephens et al., 1951, 1956). Singh and Salas (1989) reported that the mean concentration of PAN at 8 field sites, 5 of which were classified as urban, was 1.6 ppb. Maximum observed concentrations never exceeded 8 ppb. However, earlier Taylor (1969) makes reference to reports of PAN concentrations reaching peaks of 210 ppb in Los Angeles, CA. At much higher concentrations PAN has been shown to be quite toxic. The LCs0 value in mice ranges between 100 and 150 p p m depending upon exposure conditions and strain of mice used (Campbell et al., 1967). Also, PAN has been shown to cause leaf damage in plants (Taylor, 1969) as well as being a human eye irritant (Stephens, 1967). Because PAN is a reactive peroxide, it has been suspected of being mutagenic. Peak and Belser (1969) investigated reactions of PAN with isolated bacterial D N A and found that PAN can modify D N A bases; however, the reactions only occurred at p H < 5. Kleindienst et al. (1985) reported that PAN was a weak mutagen in Salmonella in TA100 regardless of whether or not rat liver $9 was used. In additional studies on several peroxyacyl nitrates, they showed that there was a linear relationship between PAN exposure duration and r e v e r t a n t s / p l a t e in TA100 without activation (Kleindienst et al., 1990). Shiraishi et al. (1986) reported that photochemical reaction products of toluene plus NO2, of which PAN was found in significant concentrations, induced sister chromatid exchanges (SCEs) in Chinese hamster V79 cells. In the only in vivo study of the genotoxicity of PAN in mammals reported to date, Heddle et al. (1993) exposed Chinese hamsters to PAN by inhalation for from 1 to 30 days at a concentration of approximately 3 ppm. Their results were inconclusive but indicated that PAN was neither mutagenic nor clastogeneic. Because PAN is a direct acting bacterial mutagen, is possibly involved in SCE induction, and is a prevalent component of polluted urban air, we investigated the in vitro and in vivo D N A damaging and cytogenetic effects of PAN on murine cells 1

Materials and methods Generation o f peroxyacetyl nitrate

Since PAN is a highly unstable species at molar concentrations and is not available commercially, it had to be synthesized in the laboratory. The technique to generate PAN employed a liquid-phase synthesis to generate the test compound in a non-volatile hydrocarbon solvent. The details o f this method have been previously described (Kleindienst et al., 1985). All reactant chemicals and reagents were from Aldrich Chemical (Milwaukee, WI). In these syntheses tridecane was used as the hydrocarbon solvent since it can be procured as a high purity chemical of extremely low volatility. ManTech Environmental Technology (Research Triangle Park, NC) synthesized and delivered the samples of PAN for these studies. Various samples of PAN were prepared by bubbling clean air at 0.5 l / m i n for 10 min through 25 ml of the P A N / t r i d e c a n e solution. The concentration in the bag was determined by taking a 10 ml sample and diluting it to 100 liters and analyzing the resulting mixture on a Columbia Scientific Industries Series 1600 Oxides of Nitrogen Analyzer (See Hudgens et al., in preparation). The bags were stored in the dark and away from heat to prevent decomposition of the PAN.

PAN treatment and cell culture In vitro experiments

Two in vitro experiments were performed. In the preliminary range finding experiment, mononuclear leukocytes from the blood of 25 male CD-1 mice (5 weeks old) were isolated on a density gradient (Erexson and Kligerman, 1987), and 7.5 × 106 cells were added to 3 ml of R P M I 1640 (Gibco, Grand Island, NY) in 10 ml ampules fitted with ampule caps. These were wrapped in

~These studies were conducted in collaboration with a series of dosimetry and mutation studies in mice exposed to PAN. Those results will be reported in a later publication.

A.D. Kligerman et al. / Mutation Research 341 (1995) 199-206

aluminum foil to prevent exposure to light and placed in a shaker at room temperature. A sample containing 3200 ppm PAN was obtained from ManTech (see above), and vials were injected with 0, 1, 5, or 10 ml of this sample (0, 0.13, 0.66, and 1.31 ~mol, respectively) after an equivalent volume or air was removed from each ampule. Controls received 5 ml of 'clean' air administered in a similar fashion. The ampules were returned to the shaker for 3 h. After treatment the cells were washed with RPMI-1640 and cultured for SCE and chromosome aberration (CA) analyses according to our standard procedures using phytohemagglutinin to stimulate mitogenesis and 5bromo-2'-deoxyuridine (BrdU), to differentiate first-, second-, and third-division metaphases by staining pattern (Erexson and Kligerman, 1987). After a total incubation time of 44 h, all cultures were harvested by centrifugation following a 3 h demecolcine (0.5 /zg/ml) (Sigma Chemical, St. Louis, MO) treatment. Cells were treated with hypotonic and fixed in 3:1 methanol/acetic acid, and slides prepared and stained using a modification of the fluorescence-plus-Giemsa method (Goto et al., 1978) using standard procedures (Erexson and Kligerman, 1987) for analyses of SCEs and CAs. A small sample of treated blood was also taken and analyzed for strand breakage using a modification of the single cell gel assay (Singh et al., 1988). Briefly, the cells were embedded in agarose on fully frosted slides as follows. A layer of 0.5% agarose was placed on the slide, a coverslip was placed on the agarose, and the slide was set on ice for 5 min. The coverslip was removed and 5 /zl of the cell suspension was diluted with phosphate buffered saline and mixed 1:1 with 1% low melting point (LMP) agarose and layered on the slide as above. A final layer of 0.5% LMP agarose was added. The cells were lysed in a high salt detergent buffer (2.5 M NaC1, 100 mM EDTA, 10 mM Tris, p H 10, 1% sodium sarcosinate, 1% Triton X-100, and 10% dimethylsulfoxide), soaked in alkaline electrophoresis buffer (30 mM N a O H l m M E D T A ) for 40 rain, and subjected to a 300 mA current at 20 volts for 20 min. The cells were digitally photographed using a chilled CCD camera (Photometrics, Tucson, Az), and the 'comet's'

201

tail length measured using IPLab ® software (Signal Analytics, (Vienna VA). Because of the toxicity observed in the first experiment, lower amounts of PAN from a sample containing 1875 ppm, were used for the second in vitro experiment. Blood was removed by cardiac puncture from 52 mice, and the mononuclear leukocytes were isolated. To each of four 10 ml ampules, 7.5 x 106 cells were added in 3 ml of RPMI-1640 and capped. Cells were exposed to 0, 1.0, 2.5, 5.0 ml of PAN (0, 0.077, 0.192, 0.384 ~mol, respectively) as described for the first experiment except that the exposure was for lh instead of 3h, and each cell pellet was washed twice with RPMI-1640 after termination of the exposure. Cultures were harvested, and slides made and stained as described above. In addition, 32 unexposed cultures were initiated for G~/S exposures. 21 h after culture initiation, the 32 cultures were pooled, the supernatant collected ('conditioned medium'), and 7.5 x 10 6 cells collected and placed in 3 ml of conditioned medium in each of four 10 ml ampules and capped with ampule caps. The ampules were injected with either 0, 0.5, 1.0, or 2.0 ml (0, 0.038, 0.077, or 0.154 /~mol) of the 1875 ppm PAN sample as previously described except that the treatment time was only 15 min. After treatment the cells were washed once in complete medium and harvested 48 h after culture initiation following a 3 h colcemid treatment. Slides were prepared as described previously. In civo experiments

A nose-only system was used for exposures (Cannon et al., 1983; Hudgens et al., in preparation) (See Hudgens et al., (in preparation) for details of its construction, testing, and exposures). Control experiments (experiments without PAN) were run before any PAN was introduced into the system. The exposure system was flushed with PAN for 30-45 min prior to the start of any mouse exposure. Male B6C3F1 mice, approximately 6 weeks of age were sedated with phenobarbital and placed 5 at a time into the inhalation apparatus. The tubes were filled with mice one at a time by the animal care technicians. As each filled mouse tube was replaced in the system, the

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202

position and time were noted on the strip chart. After an exposure time of 60 min, the mice were removed in the same order that they were placed in the system. The exposures requiring the highest concentration of PAN were performed first since the source bag was diluted with zero air to obtain lower levels of PAN. Target concentrations were 75, 40, and 10 ppm PAN, and actual concentrations achieved were 78.0, 39.2, and 15.4 ppm. The concentrations did not vary by more than 5% over the course of the run. Following exposure, the animals were anesthetized with Metofane ® (Pitman-Moore, Washington Crossing, N J) and the lungs perfused and made into a cell suspension according to a previously published procedure (Kligerman et al., 1987). After determining the viability of the cells using trypan blue (68 to 88% viable), cells from each animal's lungs were placed in a T-25 flask containing 5 ml of complete medium with 5 p.M BrdU. Complete medium consists of William's Medium E (Gibco), 10% heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 5 Izg/ml gentamicin sulfate (Schering, Kenilworth, N J). Flasks were capped loosely and placed in a 37°C incubator with a 5% C02 atmosphere. Demecolcine (0.5 /xg/ml) was added 60 h after initiation, and the cells in the flasks were trypsinized and harvested 4 h later. Cells were treated with a hypotonic KCI solution and fixed in 3:1 methanol:acetic acid, and slides prepared and stained using a modification of the fluorescence-plus-Giemsa method (Goto et al., 1978). A small sample of lung cells was prepared for analysis of single strand breakage using the SCG assay as described above. The slide preparation

for the lung cells was essentially as described above except the lung cell suspension was mixed 1:1 with the LMP agarose for the second layer without dilution. Peripheral blood leukocytes were also analyzed using the SCG assay. The blood was collected by tail snip before the lung perfusion. One or two drops of the blood from the tail were placed in a test tube containing Hank's balanced salt solution and 10% dimethylsulfoxide to prevent clotting and damage from free radicals (Ray Tice, personal communication). This suspension was centrifuged, the majority of the supernatant removed, and resuspended cells were mixed 1:1 with 1% LMP for the middle layer on the slides.

Data collection and statistical analyses

In general, for SCE and CA analyses, 25 second-division metaphases and 100 first-division metaphases, respectively, were scored from coded slides. However, in the first experiment only 50 metaphases from each of two treatments were scored for CA analysis. The replicative index (RI) was calculated from 100 consecutive metaphases by classifying them by staining pattern as either first-, second-, or third-division cells (Schneider and Lewis, 1981). Twenty-five cells from each of two slides for each treatment or animal were measured for 'tail' length in the SCG assay. For all analyses (SCE, RI, CA, SCG), a oneway analysis of variance (ANOVA) was performed with the level of significance set at 0.05. If the A N O V A was significant, a least significant difference test was used to compare the treatments to the con-

Table 1 Initial in vitro exposure of G O peripheral blood lymphocytes to PAN for 3 h PAN /zmol

Culture

Metaphases scored

Aberrant metaphases

Chrornatid deletions

Chromatid exchanges

0 0 0.13 0.13 0.66 1.31

1 50 0 2 50 4 1 50 2 2 50 2 Toxic - too few metaphases to score Toxic - too few metaphases to score

(%)

(%)

(%)

0 4 0 2

0 0 2 2

SCEs/ 25 metaphase 9.5 8.7 9.3 8.2

A.D. Kligerman et al. / Mutation Research 341 (1995) 199-206

203

Table 2 In vitro exposures of G O and G 1 / S peripheral blood lymphocytes to P A N " PAN /zmol

Culture no.

Cells scored

Aberrant cells

Chd del

Chd exch

Chm del

Chm exch

RI

SCEs/ metaphase

100 100 100 100 100 100 100 100

1 7 4 3 2 2 2 2

1 7 3 2 2 2 0 1

0 0 1 0 0 0 0 0

0 0 0 0 0 0 0 1

0 0 0 1 0 0 0 0

1.16 1.15 1.46 1.48 1.47 1.45 1.32 1.48

7.24 8.84 9.12 7.60 7.60 7.36 8.64 8.16

G l / S Exposures for 15 min 0 1 100 0 2 100 0.038 1 100 0.038 2 100 0.077 1 100 0.077 2 100 0.154 1 100 0.154 2 100

3 2 2 1 2 1 3 1

3 2 1 1 2 1 3 1

0 0 0 0 0 0 0 0

0 0 1 0 0 0 0 0

0 0 0 0 0 0 0 0

1.32 1.34 1.28 1.31 1.27 1.27 1.32 1.21

9.28 9.24 11.16 10.16 9.80 9.40 9.00 8.28

G o Exposures for 1 h 0 1 0 2 0.077 1 0.077 2 0.192 1 0.192 2 0.384 1 0.384 2

a Abbreviations: Chd del, chromatid deletion; Chd exch, chromatid exchange; C h m del, chromosome deletion; C h m exch, chromosome exchange; RI, replicative index.

trol using a one-tailed test with 0.05 as the level of significance.

SCE or CA induction was evident although at the lowest concentration of PAN examined in the G 1 / S , a small statistically significant increase in SCE was observed (Fig. 2A and 2B). In addition,

Results and discussion

While P A N was quite toxic both in vitro and in vivo, it produced little if any genotoxic effects in the assays used. In the preliminary in vitro experiment (Table 1), G O PBLs were exposed to either 0, 0.13, 0.66, or 1.31 /zmol of PAN. The two highest exposures proved too toxic for cytogenetic analyses (i.e., too few metaphases); whereas the lowest concentration gave no evidence of SCE or CA induction (Fig. 1A). The SCG assay showed a statistically significant increase in 'tail' length indicating D N A strand breakage or alkaline-labile sites (Fig. 1B). However, this might have been due to nonspecific strand breakage caused by cell death a n d / o r lysis. In the second in vitro experiment (Table 2), PBLs were exposed to aliquots of P A N up to 0.38 ~ m o l in a G O exposure or to 0.15/zmol in a G 1 / S exposure. No concentration-related indication of

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204

A.D. Kligerman et al. / Mutation Research 341 (1995) 199-206

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exposures to P A N give scant evidence that it can directly damage D N A at concentrations compatible with cell survival. The in vivo studies (Table 3) provide addi-

for the G O exposure, P A N appeared to cause a statistically significant increase in the RI, indicating a quickening of the cell cycle. This was not seen in the G ~ / S exposure. Thus, the in vitro Table 3 Cytogenetic effects in murine lung cells of PAN inhalation a PAN [ppm]animal #

Percent aberrant cells

Chd del per 100 cells

Chd exch per 100 cells

Chm del per 100 cells

Chm exch per 100 cells

RI

SCEs/ metaphase

0-1 0-2 0-3 0-4

14 29 7 14

12 31 5 16

6 15 7 9

1 0 0 1

0 0 0 1

2.40 1.94 2.38 2.25

19.5 22.3 15.8 18.4

15-1 15-2 15-3 15-4

9 10 13 25

11 7 11 37

5 3 9 25

3 0 0 1

0 0 0 1

2.22 2.18 2.29 1.46

14.8 16.1 18.5 18.7

39-1 39-2 39-3 39-4

13 15 20 11

18 16 18 18

15 14 23 10

0 1 1 0

0 0 0 0

2.06 2.21 2.01 2.17

21.8 17.1 19.1 16.2

78-1 78-2 78-3 78-4

13 13 13 6

15 12 8 5

7 9 3 6

1 2 2 0

0 0 0 0

2.23 2.19 2.48 2.51

16.0 14.0 16.0 13.8

a Abbreuiations: Chd del, chromatid deletion; Chd exch, chromatid exchange; Chm del, chromosome deletion; Chm exch, chromosome exchange; RI, replicative index.

A.D. Kligerman et al. / Mutation Research 341 (1995) 199-206

205

P~-s

80

A

LUNGCELLS

30

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25

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78

PAN CONCENTRATIONS [ppm]

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78

PAN CONCENTRATIONS [ppm]

Fig. 3. In vivo nose-only inhalation study with PAN. (A) Sister chromatid exchange and chromosome aberration analyses in cultured lung cells of exposed mice. (B) Single cell gel electrophoresis assay in peripheral blood lymphocytesand lung cells. (Error bars represent the standard deviation of the mean.)

tional evidence that PAN is not a potent genotoxicant as measured by SCEs or CAs (Fig. 3A) or D N A strand breakage (Fig. 3B). Exposure to near toxic concentrations of P A N for 1 h caused no statistically significant differences between control and treated mice with regards to SCE, CA, and single strand D N A damage to their lung cells. Also, no evidence was found that PAN increased D N A damage, as measured by the SCG assay in the PBLs of the exposed mice, and no statistically significant changes were observed in the cell cycle kinetics of the lung cells (Table 3). Recently, Heddle et al. (1993) published data from their studies in which Chinese hamsters were exposed to PAN by inhalation (whole-body, rather than nose-only exposure) at approximately 3 p p m for from 1 to 30 days. They then examined the mutant frequency in cultured lung cells as well as the incidence of micronuclei in cultured lung cells and bone marrow and peripheral blood polychromatic erythrocytes from control and exposed animals. Although they found several instances of elevated mutant frequencies in various experiments, overall the results were statistically insignificant. They could not 'determine unambiguously whether or not PAN is mutagenic in

vivo'. Similarly, both MN assays failed to demonstrate statistically significant clastogenicity that could be attributed to PAN exposure. Their in vivo results coupled with our in vitro and in vivo results indicate that PAN is not a potent point mutagen, clastogen, or by inference an aneugen, if P A N reached the bone marrow, in mammalian cells. They also may indicate that the SCE induction seen by Shirashi et al. (1986) in V79 cells exposed to photochemical reaction products of toluene and N O 2 may not be due to the PAN formed. Although these results are in contrast to the bacterial mutagenicity results, it must be r e m e m b e r e d that PAN is a relatively weak bacterial mutagen, and it is more than likely that bacteria can survive and multiply at concentrations of PAN that are toxic to mammalian cells.

Acknowledgements The authors would like to thank M. Kohan, P. Kwanyuen, and M. Bryant for their technical expertise and assistance; L. Claxton, E. Hudgens, T. Kleindienst, and J. Lewtas for thoughtful dis-

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A.D. Kligerman et al. / Mutation Research 341 (1995) 199-206

cussions and suggestions during the course of the study and preparation of the manuscript; and D. Walsh with help in coordinating the project. K. Mottus was supported by training grant No. 5T32ES07126.

References Campbell, K.I., G.L. Clarke, L.O. Emik and R.L. Plata (1967) The atmospheric contaminant peroxyacetyl nitrate, Arch. Environ. Health, 15, 739-744. Cannon, W.C., E.F. Blanton and K.E. McDonald (1983) The flow-past chamber: an improved nose-only exposure system for rodents, Am. Ind. Hyg. Assoc. J., 44, 923-928. Erexson, G.L. and A.D. Kligerman (1987) A modified mouse peripheral blood lymphocyte culture system for cytogenetic analysis, Environ. Mol. Mutagen., 10, 377-386. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in differential staining of sister chromatids, Chromosoma, 66, 351-359. Heddle, J.A., P.B. Shepson, J.D. Gingerich and K.W. So (1993) Mutagenicity of peroxyacetyl nitrate (PAN) in vivo: tests for somatic mutations and chromosomal aberrations, Environ. Mol. Mutagen., 21, 58-66. Hudgens, E.E., T.E. Kleindienst and M. Kohan (in preparation) Development of an exposure system for volatile organic compounds: exposures of rodents to radiolabeled 3H-PAN. Kleindienst, T.E., P.B. Shepson, E.O. Edney and L.D. Claxton (1985) Peroxyacetyl nitrate: measurement of its mutagenic activity using the Salmonella/mammalian microsome reversion assay, Mutation Res., 157, 123-128. Kleindienst, T.E., P.B. Shepson, D.F. Smith, E.E. Hudgens, C.M. Nero, L.T. Cupitt, J.J. Bufalini and L.D. Claxton (1990) Comparison of mutagenic activites of several peroxyacyl nitrates, Environ. Mol. Mutagen., 16, 70-80.

Kligerman, A.D., J.A. Campbell, G.L. Erexson, J.W. Allen and M.D. Shelby (1987) Sister chromatid exchange analysis in lung and peripheral blood lymphocytes of mice exposed to methyl isocyanate by inhalation, Environ. Mutagen. 9, 29-36. Peak, M.J. and W.L. Belser (1969) Some effects of the air pollutant, peroxyacetyl nitrate upon deoxyribonucleic acid and upon nucleic acid bases, Atmos. Environ., 3, 385-397. Schneider, E.L. and J. Lewis (1981) Aging and sister chromatid exchange. VIII. Effect of aging environment on sister chromatid exchange and cell cycle kinetics in Erlich ascites tumor cells. A brief note, Mech. Ageing Dev., 17, 327-330. Shiraishi, F., S. Hashimoto and H. Bandow (1986) Induction of sister°chromatid exchanges in Chinese hamster V79 cells by exposure to the photochemical reaction products of toluene plus NO 2 in the gas phase, Mutation Res., 173, 135-139. Singh, H.B. and L.J. Salas (1989) Measurements of peroxyacetyl nitrate (PAN) and peroxypropionyl nitrate (PPN) at selected urban, rural and remote sites, Atmos. Environ., 23, 231-238. Singh, N.P., M.T. McCoy, R.R. Tice and E.L. Schneider (1988) A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res., 175, 184-191. Stephens, E.R. (1967) The formation, reactions and properties of peroxyacyl nitrates (PANs) in photochemical air pollution, Adv. Environ. Sci. Technol., 1, 119-146. Stephens E.R., E.F. Darley, E.C. Taylor and W.E. Scott (1951) Photochemical reaction products in air pollution, Int. J. Air Wat. Pollut., 4, 79-100. Stephens, E.R., W.E. Scott, P.L. Hanst and R.C. Doerr (1956) Recent developments in the study of the organic chemistry of the atmosphere, J. Air Pollut. Control Assoc., 6, 159165. Taylor, O.C. (1969) Importance of peroxyacetyl nitrate (PAN) as a phytotoxic air pollutant, J. Air Pollut. Control Ass., 19, 347-351.