Human cell mutagenicity of mono- and dinitropyrenes in metabolically competent MCL-5 cells

Genetic Toxicology

ELSEVIER

Mutation Research 322 (1994) 233-242

Human cell mutagenicity of mono- and dinitropyrenes in metabolically competent MCL-5 cells William F. Busby Jr. a,,, Bruce W. Penman h, Charles L. Crespi b a

Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA b Gentest Corporation, Woburn, MA 01801, USA

Received 1 December 1993; revised 17 March 1994; accepted 11 April 1994

Abstract Nitropyrenes are ubiquitous environmental pollutants that may pose a human health hazard because some are highly potent mutagens and carcinogens. The mutagenicity (trifluorothymidine resistance at the thymidine kinase locus) of 1-, 2-, and 4-nitropyrene (1-, 2-, and 4-NP), 1,3-, 1,6-, and 1,8-dinitropyrene (1,3-, 1,6-, and 1,8-DNP), and pyrene was assessed in a quantitative forward mutation assay using a metabolically competent line (MCL-5) of human B-lymphoblastoid cells. These cells contain endogenous cytochrome P450 activity (CYP1A1) and two plasmids that express cDNAs for four additional P450s (CYP1A2, CYP2A6, CYP2E1, CYP3A4) and microsomal epoxide hydrolase found in human liver. The major finding is that 2-NP and 1,3-DNP, both potent bacterial mutagens, were nonmutagenic in this assay. The following mutagenic potency series, expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml, was obtained: 1,6-DNP (0.8), 1,8-DNP (1.5), 4-NP (3.1), 1-NP (9.1), 2-NP ( > 81), 1,3-DNP ( > 86), pyrene ( > 494). There was over an ll-fold difference between the most potent (1,6-DNP) and the least potent (1-NP) mutagen. 1,6-DNP was approximately twice as mutagenic as 1,8-DNP, which was almost twice as mutagenic as 4-NP, which, in turn was nearly three times as potent as 1-NP. This is the first report on the testing of 2-NP and 4-NP for mutagenicity in mammalian cell cultures. The human cell mutagenicity of these compounds was discussed in terms of potency series of nitropyrenes obtained from animal carcinogenicity experiments and other mammalian cell mutagenicity assays. Keywords: Mononitropyrene; Dinitropyrene; Mutagenicity; Human cell; MCL-5 cell

1. Introduction

Abbreviations: DMSO, dimethyl sulfoxide; 1,3-DNP, 1,3dinitropyrene; 1,6-DNP, 1,6-dinitropyrene; 1,8-DNP, 1,8-dinitropyrene; MDMC, minimum detectable mutagen concentration; 1-NP, 1-nitropyrene, 2-NP, 2-nitropyrene; 4-NP, 4-nitropyrene; PAH, polycyclic aromatic hydrocarbons. * Corresponding author.

N i t r o p y r e n e s a r e a g r o u p of e n v i r o n m e n t a l p o l l u t a n t s t h a t a r e p r e s e n t in emissions f r o m t h e c o m b u s t i o n of fuels a n d o t h e r o r g a n i c m a t e r i a l s ( I A R C , 1989), o r t h a t a r e f o r m e d in the a t m o s p h e r e f r o m the r e a c t i o n o f p y r e n e with hydroxyl radicals a n d N O 2 o r with N O 3 radicals (Pitts et al., 1978; Pitts, 1987). S u b s t a n t i a l r e s e a r c h has

0165-1218/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1218(94)00030-7

234

W.F. Busby et al. / Mutation Research 322 (1994) 233-242

been directed towards nitropyrenes because some of these compounds are potent mutagens in bacteria and carcinogens in rats and mice (Rosenkranz and Mermelstein, 1985; Tokiwa and Ohlfishi, 1986; IARC, 1989). 1-NP is the most abundant nitropyrene in combustion and atmospheric samples (Rosenkranz and Mermelstein, 1983; 1985). It was detected in most extracts of atmospheric particles collected from urban and rural sites (Tokiwa et al., 1983; Gibson, 1986; Ramdahl et al., 1986), and was associated with emissions from diesel engines (Paputa-Peck et al., 1983), domestic kerosene heaters, and manufactured gas and propane flames (Tokiwa et al., 1985: Kinouchi et al., 1988). 1-NP was mutagenic to bacteria and some cultured rodent cells and was carcinogenic in rats and mice (IARC, 1989). It also induced mutations, DNA damage, and transformation in cultured human cells. 2-NP and 4-NP were found in air particle samples, but their presence in combustion emissions has not been confirmed (Ramdahl et al., 1986; Korfmacher et al., 1987). These compounds presumably originated as atmospheric transformation products of pyrene (Atkinson et al., 1990). Although 2-NP has been reported to be a strong bacterial mutagen in the Ames reversion assay in both the presence and absence of rat liver supernatant (Hirayama et al., 1988; Yu et al., 1992), it was active only with liver supernatant in the Salmonella TM677 forward mutation assay (Busby et al., 1994). 2-NP was weakly carcinogenic when injected into rats (Imaida et al., 1991a). 4-NP, a strong bacterial mutagen (Fu et al., 1985; Busby et al., 1994), was a potent inducer of mammary tumors in rats (Imaida et al., 1991a) and lung and liver tumors in mice (Wislocki et al., 1986). The three dinitropyrene isomers (1,3-, 1,6-, and 1,8-DNP) were consistently found at low concentrations (10- to 100-fold lower than 1-NP) in ambient air (Tokiwa et al., 1983; Gibson, 1986), diesel exhaust particles (Paputa-Peck et al., 1983), and emissions from combustion of kerosene, propane, and manufactured gas (Tokiwa at al., 1985; Kinouchi et al., 1988). In general, the minor contributions of DNPs to the environmental load of nitropyrenes is offset by their high biological

potency. DNPs are among the most potent bacterial mutagens known with potencies 2-4 orders of magnitude greater than those for mononitropyrenes (Rosenkranz and Mermelstein, 1985; Busby et al., 1994). 1,6- and 1,8-DNP also induced DNA damage, chromosomal abnormalities, and mutations in mammalian cells and tumors in rats and mice (IARC, 1989; Imaida et al., 1991b; Sawada et al., 1991). In a program to identify the most important human cell mutagens in ambient air we examined the comparative mutagenicity of selected monoand dinitropyrenes, known to be present in the atmosphere, at the thymidine kinase (tk) locus in a quantitative mutation assay using e metabolically competent human lymphoblastoid B cell line (MCL-5 cells). These cells were derived from an L3 variant (Davies et al., 1989) of AHH-1 TK + / - cells, which contained the cytochrome P450 CYP1A1 (Crespi and Thilly, 1984), by transfecting two plasmids (pME23 and pH441) that stably expressed cDNAs for human microsomal epoxide hydrolase and four additional cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4) found in human liver (Crespi et al., 1991). Classes of compounds demonstrated to be mutagenic in this cell line included certain PAH, aromatic amines, and nitrosamines. The PAH benzo[a]pyrene and 3-methylcholanthrene were particularly potent and were active at concentrations < 10 ng/ml. However, fluoranthene and cyclopenta[cd]pyrene were not mutagenic in MCL-5 cells (data not shown), presumably because these PAH were either not activated by the cytochromes present or were deactivated by the presence of epoxide hydrolase.

2. Materials and methods

2.1. Test chemicals

Compounds for testing were obtained from the following sources: recrystallized pyrene (97.6%) (CAS No. 129-00-0) from Dr. R. Colin Garner (University of York Cancer Center, UK); 1-NP (99.8%) (5522-43-0) from AccuStandard Inc., New Haven CT; 2-NP ( > 95%) (789-07-1) from Chem-

W.F. Busby et al. / Mutation Research 322 (1994) 233-242

syn Science Laboratories, Lenexa KS and from Profs. J. Cornelisse and J. Lugtenburg (Leiden University, The Netherlands); 4-NP (> 99%) (57835-92-4) also from Chemsyn; 1,6-DNP (98%) (42397-64-8) and 1,8-DNP (98%) (42397-65-9) from Aldrich Chemical Co., Milwaukee WI; and 1,3-DNP ( > 99%) (75321-20-9) from Sigma Chemical Co., St. Louis, MO. 2.2. Cell culture chemicals

Donor horse serum was purchased from JRH Biosciences (Kansas City MO); hygromycin B from Calbiochem (San Diego CA); L-histidinol dihydrochloride from Biosynth International (Skokie, IL), and dimethylsulfoxide (DMSO) from J.T. Baker (Phillipsburg, N J). All other chemicals were obtained from Sigma (St. Louis, MO). 2.3. Mutation assays

The mutagenicity of test compounds was assessed at the thymidine kinase (tk) locus in MCL-5 cells that were transfected with plasmids expressing cDNAs for human cytochrome P450s and epoxide hydrolase (Crespi et al., 1991). Stock cultures were passaged every other day to 2.5 x 105 cells/ml or every third day to 1 x 105 cells/ml in RPMI 1640 medium without histidine and supplemented with 9% (v/v) horse serum. The medium also contained 2 mM L-histidinol and 100 (2 day passage) or 200 (3 day passage)/xg/ml of hygromycin B to maintain selection for the plasmids and 20 ~ g / m l of delta-aminolevulinic acid to support heme synthesis. All incubations were done in a humidified 37°C incubator with a 5% CO 2 atmosphere. Before use in a mutation assay, exponentially growing MCL-5 cells were grown for 3 days in medium containing HAT (2 x 10 - 4 M hypoxanthine, 8 x 10 -7 M aminopterin, 3.5 X 10 -5 M thymidine) to remove pre-existing mutants. The cells were diluted to 2.5 x 105 cells/ml after one day. Following HAT treatment, the cells were centrifuged (1000 x g for 5 min) and resuspended at 4-5 x 105 cells/ml in medium containing thymidine and hypoxanthine (TH). The cultures were passaged one day after resuspension with

235

normal medium (without TH) and scaled up depending on the number of cells required for the experiment. The cells were treated with test substance 3-4 days after initial resuspension in THcontaining medium. Normal RPMI (with histidine) containing 100 U / m l penicillin and 100/xg/ml streptomycin sulfate was used for all subsequent steps. In the mutagenicity assay 6 x 106 cells per 12 ml replicate culture (25 cm 2 flask) were exposed to appropriate concentrations of test substance (delivered in 60 /zl DMSO or less) for 28 h. Each substance was tested in at least two separate experiments with replicate cultures and replicate positive (1 /xg/ml benzo[a]pyrene) and negative (DMSO) controls also were run with every experiment. Following treatment the cultures were centrifuged and resuspended in fresh media (20 ml in a 75 cm 2 T-flask), and allowed to grow for 3 days. After the first day the cultures were counted and diluted with fresh medium to 2 x 105 cells/ml. The serum supplement for this dilution contained two-thirds heat-treated serum (1 h at 56°C). After this 3-day phenotypic expression period, cultures were plated in the presence (in three 96-well microtiter plates, 20,000 cells/well) and absence (in two 96-well plates at 2 cells/well) of 4 /zg/ml trifluorothymidine, incubated for 13 days, and the plates scored for the presence of a colony in each well. The serum supplement for plating was heat treated. Toxicity of the test compounds was estimated by measuring the cumulative growth of the cultures from the beginning of treatment until plating. Relative survival was calculated by dividing the growth of the treated cultures by the cumulative growth of the negative control cultures. The plating efficiency and observed mutant fraction were calculated for each culture from the fraction of wells without colonies using the Poisson distribution (Furth et al., 1981). The mean mutant fraction for each test condition was compared to the concurrent (Dunnett's t-test, p = 0.05) and historical (99% upper confidence limit calculated on the basis of a Gamma distribution and corrected for the number of treatment concentrations) negative control observations (Penman and

W.F. Busby et al. /Mutation Research 322 (1994) 233-242

236

Crespi, 1987). The test substance was designated mutagenic only when both statistical tests were positive. Mutagenicity was expressed as the MDMC, the concentration of test substance where the dose-response plot intercepted the 99% upper confidence limit for the historical negative control. Each experiment, consisting of duplicate concentrations of test samples in four dose groups, was performed at least twice on different days.

03

~o.5

n.-

o

i

30

'O

I

i

I//-

-'~

I

I

0 Pyrene

E/

"

I

I l0

2.P

= 20

3. Results

o

"6

The MCL-5 human cell assay was employed to measure mutagenicity at the thymidine kinase (tk) locus for several biologically active mononitropyrenes (1-, 2-, and 4-NP) and dinitropyrenes (1,3-, 1-6-, and 1,8-DNP), as well as the parent P A H pyrene (Fig. 1). Simultaneous determinations of toxicity and mutagenicity were made for each culture, thus the number of mutant ceils could be expressed quantitatively as the mutant fraction in surviving cells. In this series of experiments the mean _+ SD for the mutant fraction of the D M S O solvent control was 2.19 + 0.81 and 70.9 +_ 19.9 for the positive control (100 n g / m l of benzo[a]pyrene). A 99% upper confidence limit for the historical control mutant fraction for MCL-5 ceils under the conditions of this assay was calculated as 5.3 x 10 -6 based upon 83 experiments. Dose-response plots for the mutagenicity and toxicity of pyrene and the mononitropyrenes are shown in Fig. 2. Pyrene was clearly nonmutagenic at concentrations up to 100 p~g/ml, however, some toxicity was apparent with relative survival reduced by 25%. 2-NP from two different sources

tO

1

3 8 7 6

5

Fig. 1. Structure and numbering system of pyrene.

o [a-

~Olo

0-1 0

~

I//I

20"25

I

I

50

75

100

Concentrofion ( u g / m l ) Fig. 2. Toxicity (upper panel) and mutagenicity (lower panel) of pyrene (o) and the mononitropyrenes 1-NP (o), 2-NP (zx),

and 4-NP ( • ) in MCL-5 cells. The 99% upper confidence limit for the historical negative control mutant fraction of 5.3X10 -6 is represented by the dashed line. Results are presented as the mean + SD.

was also nonmutagenic to MCL-5 cells at up to 20 ~ g / m l with little or no evidence of toxicity over this range of concentrations. By contrast, 4-NP was the most mutagenic of the mononitropyrenes with an M D M C (the intercept of the dose-response plot with the historical 99% upper confidence limit with a mutant fraction of 5.3 x 10 - 6 ) of 0 . 8 / z g / m l and a nearly linear 15-fold increase in the mutant fraction up to 1 0 / ~ g / m l . Relative survival decreased by over 30% over this concentration range. For these and subsequent experiments, relative survival data were sufficiently reproducible to preclude the use of error bars in the figures. Combined data from three experiments performed in replicate over the concentration range of 1 - 6 / x g / m l indicated that 1-NP was mutagenic only at 4 /~g/ml, where the mutant fraction was nearly 4-fold higher than the concurrent back-

W.F. Busby et al. / Mutation Research 322 (1994) 233-242

ground (Fig. 2). This was the only concentration where the mutant fraction was significantly different from both the concurrent or historical control, the criteria by which a positive response is determined. The testing of 1-NP was limited to these concentrations because of solubility and the maximum amount of D M S O (0.5%) tolerated by the cells. No differences were noted from the concurrent controls at any other concentration; however, there was a significant difference from the historical control value at 1 /.~g/ml. There was a dose-dependent 25% decrease in survival in this experiment. Taken together, these data showed that 1-NP was a borderline mutagen in this assay. Our experience has been that the source, and thus the purity, of 1-NP plays a major role in the mutagenic response obtained in MCL-5 cells. The dose-response plot for 1-NP from an-

I

i

i

i

i

i

M / / i

i

I

i

I

I

I

~ i.o w

O~

•

>~o5

~

o

I

I

I

I

I//I

o 1,3-DNP • 1,6-DNP A 1,8- DNP

30 I ~0 x

g ~ 2O

o

:~o

w

0

I

I

I

2

I

I

I //

I

.3 4 5 I0 Concentrotion ( u g / m l

i

I

I

20

Fig. 3. Toxicity (upper panel) and mutagenicity (lower panel) of the dinitropyrenes 1,3-DNP (©), 1,6-DNP (e), and 1,8-DNP ( A ) in MCL-5 cells. The dashed line represents the 99% upper confidence limit for the historical negative control mutant fraction (5.3x10-6). Results are presented as the mean _+SD.

237

Table 1 Comparative mutagenicity of mono-and dinitropyrenes in MCL-5 cells Compound

MDMC a (nmol/ml)

Pyrene 1-NP 2-NP 4-NP 1,3-DNP 1,6-DNP 1,8-DNP

> 494 9.1 > 81 3.1 > 86 0.8 1.5

a Concentration of compound at which the dose-response plot intersected the 99% upper confidence limit for historical negative controls (horizontal dashed line in Figs. 2 and 3 at a mutant fraction of 5.3 × 10 6).

other commercial supplier (listed as 97% pure) was linear over the range of concentrations tested ( 1 - 6 izg/ml) and gave a maximum mutant fraction of over 9 times higher than the concurrent control value (data not shown). The human cell mutagenicity of the dinitropyrenes is illustrated in Fig. 3. 1,6- and 1,8-DNP were potent human cell mutagens, exceeding the M D M C at the lowest concentration (0.5 t z g / m l ) tested. The dose-response plot for 1,6-DNP was linear to 1 / z g / m l , and that for 1,8-DNP linear to 2.5 ~ g / m l . However, 1,3-DNP was nonmutagenic at concentrations up to 25 /zg/ml. 1,6-DNP was the more toxic of the three isomers (70% relative survival at 5 izg/ml). Survival was 80-90% for the 1,8- and 1,3- isomers at the same concentration. A comparison of the relative mutagenic potencies of the nitropyrenes calculated as the M D M C s expressed on a molar concentration basis is summarized in Table 1. Pyrene, 2-NP, and 1,3-DNP were inactive in this assay up to the maximum concentrations tested. 1,6-DNP was the most mutagenic nitropyrene and was approximately twice as potent as 1,8-DNP, which in turn was nearly twice as active as 4-NP. 1-NP was about three times less mutagenic than 4-NP. Thus, the range of the M D M C s for the mutagenic nitropyrenes varied by about a factor of 11 from the most potent (0.8 n m o l / m l for 1,6-DNP) to the least potent (9.1 n m o l / m l for 1-NP).

238

W.F. Busby et al. / Mutation Research 322 (1994) 233-242

4. Discussion

The mutagenicity of several, environmental mono- and dinitropyrenes was measured in a quantitative forward mutation assay using a line of metabolically-competent human lymphoblastoid cells (MCL-5 cells). These data include the first report of the mutagenicity of 2-NP and 4-NP in mammalian cells. The following mutagenic potency series, expressed as the M D M C in n m o l / m l , was obtained: 1,6-DNP (0.8), 1,8-DNP (1.5), 4-NP (3.1), 1-NP (9.1), 2-NP ( > 81), 1,3-DNP ( > 86), pyrene ( > 494). The human cell potency series differed markedly from that reported for Salmonella typ h i m u r i u m . In the absence of rat liver supernatant bacterial assays were much more sensitive to most nitropyrenes than the MCL-5 cell assay and did not discriminate among the dinitropyrene isomers. For example, 1,3-, 1,6-, and 1,8-DNP typically produced 1-3 × 105 m u t a n t s / n m o l e in reversion assays (Rosenkranz and Mermelstein, 1985; Tokiwa and Ohnishi, 1986) or were mutagenic at concentrations as low as 0.5-2 p m o l / m l in a forward mutation assay without metabolic activation (Busby et al., 1994). In contrast to bacteria, 1,3-DNP was nonmutagenic in MCL-5 cells and the M D M C s for 1,6- and 1,8-DNP were about three orders of magnitude higher. There was greater consistency when results from the MCL-5 cell assay were compared to the bacterial assays performed in the presence of rat liver supernatant. Because the 2 - 3 orders of magnitude increase in the mutagenicity of dinitropyrenes was eliminated when liver supernatnat was added, there was only a 6-fold difference in activity between the most potent of the dinitropyrenes (1,6-DNP) and the least potent of the mutagenic mononitropyrenes (1-NP) in the bacterial forward mutation assay (Busby et al., 1994). This finding was more consistent with the results from the human cell assay where the difference was about l 1-fold between the same two compounds, notwithstanding the fact that 2-NP was nonmutagenic in MCL-5 cells. Furthermore, the M D M C s for the mutagenic nitropyrenes ranged between 1-9 n m o l / m l for the two assays. The human cell potency series appears to be in

concordance with recent carcinogenicity studies in rodents where several nitropyrenes were concurrently tested with the same experimental protocol. 1,6- and 1,8-DNP were strong carcinogens and induced injection site tumors (histiocytomas) that caused premature death in 90-100% of the rats (Imaida et al., 1991b). As in the MCL-5 assay, 1,6-DNP was the more potent of the two isomers with an average survival time of the treated animals 40% less than that of 1,8-DNPtreated animals. Neither control rats nor those treated with equivalent amounts of 1-NP or 1,3D N P developed tumors of this type. Consequently, these animals survived until the end of the experiment or 2 - 4 times longer than the dinitropyrene-treated rats. However, 1-NP and 1,3-DNP did cause a significant increase in animals with m a m m a r y tumors. Injection site tumors were also induced by 1,8-DNP, but not 1,3-DNP, following subcutaneous injection into mice (Otofuji et al., 1987). In a companion study in which rats were treated with a larger dose of mononitropyrenes, but where the experiment was terminated four months earlier, m a m m a r y tumors were found in 60-80% of rats treated with 4-NP (Imaida et al., 1991a). Neither 1-NP nor 2-NP induced mammary tumors under these conditions, although leukemia was found in over 20% of the rats treated with 2-NP. Taken together these data affirmed the potency of 1,6-DNP, 1,8-DNP, and 4-NP as human cell mutagens and animal carcinogens, as well as the comparatively weak activity or lack of potency for 1,3-DNP, 1-NP, and 2-NP. Pyrene was not tested in any of these studies, but the International Agency for Research on Cancer (IARC) concluded that there was no evidence for its carcinogenicity based primarily on several skin painting studies in mice (IARC, 1983). There is only limited information on the mutagenic or genotoxic responses of nitropyrenes reported for other human cell lines. H u m a n foreskin diploid fibroblasts were transformed (Howard et al., 1983) and mutated at the hgprt locus (Patton et al., 1986) by 10-50 uM 1-NP. Mutations (hgprt locus) and unscheduled D N A synthesis were also induced by 1-NP in H e p G 2 cells, a

W.F. Busby et al. / Mutation Research 322 (1994) 233-242

human hepatoma cell line (Eddy et al., 1987). In contrast to the order of potency in the MCL-5 assay, 1,6- and 1,8-DNP were inactive and 1,3DNP had limited activity for both these genotoxic endpoints in HepG2 cells (Eddy et al., 1986). Neither 1,3-DNP nor 1,6-DNP induced micronuclei formation in HepG2 cells at 3 nmol/ml, the only concentration tested (Roscher and Wiebel, 1992). The mutagenic potency series in MCL-5 cells more closely resembled responses obtained with rodent cell cultures. 1-NP was consistently nonmutagenic and 1,6- and 1,8-DNP were strongly mutagenic at different loci in several Chinese hamster cell lines in the absence of rat liver supernatant (Nakayasu et al., 1982; Li and Dutcher, 1983; Takayama et al., 1983; Fifer et al., 1986). In those instances where it was tested, 1,3-DNP was either inactive or only weakly mutagenic. At present there is no information on the activity of 2-NP and 4-NP in other mammalian cell cultures. Results from the MCL-5 assay were not necessarily in agreement with potency series obtained for certain genotoxic endpoints other than mutagenicity in rodent cell cultures, such as sister chromatid exchanges in the absence of rat liver supernatant (Nachtman and Wolff, 1982), unscheduled DNA synthesis (Mori et al., 1987), or single-strand DNA breaks (MOiler and Thorgeirsson, 1985). The variations in responses of these biological systems, including human cells (Patton et al., 1986), to nitropyrenes likely reflect differences in metabolic activation processes by which these compounds are transformed into DNA-reactive intermediates. Nitroreduction a n d / o r ring oxidation are two pathways implicated in nitropyrene activation (Djuri6 et al., 1988; Roy et al., 1989; Smith et al., 1990; Thornton-Manning et al., 1991). The enzymatic complement of nitroreductases may be one basis for the different responses of cultured cells to nitropyrenes. As opposed to bacteria, most mammalian cell lines (including MCL-5 cells) were only weakly mutated or not mutated by 1-NP or 1,3-DNP, but were highly sensitive to 1,6- and 1,8-DNP (Eddy et al., 1986; 1987). Human hepatoma (HepG2) cells responded in the opposite manner. This may be

239

because 1-NP and 1,3-DNP are reduced by a nitroreductase that transfers a single electron, where as 1,6- and 1,8-DNP are reduced by an enzyme that transfers 2 electrons (Eddy et al., 1986). This differential sensitivity could reflect the relative balance between the activities of these two enzymes. Cytochrome P450s have been implicated in the biological activity and both the reductive and oxidative metabolism of nitropyrenes. A severalfold reduction of dinitropyrene-induced chromosomal aberrations in a subclone of Chinese hamster lung fibroblasts was correlated with a 50% reduction in cytochrome P450 reductase activity (Sawada et al., 1991). Addition of various rat liver cytochrome P450s to a reconstituted system containing rat liver cytochrome P450 reductase caused large increases in the reduction of 1-NP to 1-aminopyrene (Saito et al., 1984), and oxidized metabolites of 1-NP were recovered from incubation mixtures of rabbit liver cytochrome P450s, P450 reductase, and epoxide hydrolase (Howard et al., 1988). A role for human cytochrome P450s in nitropyrene metabolism was demonstrated in studies with human hepatoma (HepG2) cells containing cDNAs for twelve individual human P450s (Howard et al., 1990). Of the P450s present in MCL-5 cells, only CYP2A6 (then known as CYP2A3) and CYP3A4 significantly metabolized 1-NP to produce a variety of ring oxidized products. CYP1A2 and CYP2E1 did not significantly metabolize 1-NP, and CYP1A1 was not included in this study. Incubation of 1,3-, 1,6-, and 1,8-DNP with microsomes from different human liver samples showed that these compounds were inactivated (i.e. metabolized in an unspecified manner) to varying degrees as measured by loss of their ability to produce DNA damage in Salmonella (Shimada and Guengerich, 1990). Correlation analysis and immunoinhibition studies indicated that the three dinitropyrenes were inactivated by different enzymes. The inactivation of 1,6-DNP was correlated with CYP3A4 activity and the inactivation of 1,3-DNP was linked to CYP1A2 (and possibly CYP1A1) activity. Our data suggest that the MCL-5 human cell assay provides a sensitive and cost-effective means

240

W.F. Busby et aL /Mutation Research 322 (1994) 233-242

to assess the mutagenicity of other nitro-PAH. As an added bonus, the relevance of nitropyrene exposure to human health can be examined further because differences in the defined complement of cytochrome P450s in MCL-5 cells and other available cell lines permits relating biological activity to individual human cytochrome P450s and to specific metabolic pathways operative in humans. Studies are in progress to measure and compare the mutagenicity of other environmentally relevant nitro-PAil with their parent PAIl.

Acknowledgements We thank Xia He for the preparation of samples for testing and the technical staff of Gentest Corporation for performing the human cell mutation assay. This research was supported by Grant No. P01-ES01640 from the National Institute of Environmental Health Sciences.

References Atkinson, R., J. Arey, B. Zielinska and S.M. Aschmann (1990) Kinetics and nitro-products of the gas-phase OH and NO 3 radical-initiated reactions of naphthalene-ds, fluoranthene-d10, and pyrene, Intern. J. Chem. Kinetics, 22, 9991014. Busby, W.F., Jr., H. Smith, W.W. Bishop and W.G. Thilly (1994) Mutagenicity of mono- and dinitropyrenes in the Salmonella typhimurium TM677 forward mutation assay, Mutation Res., 322, 221-232. Crespi, C.L. and W.G. Thilly (1984) Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism, Mutation Res., 128, 221-230. Crespi, C.L., F.J. Gonzalez, D.T. Steimel, T.R. Turner, H.V. Gelboin, B.W. Penman and R. Langenbach (1991) A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: Application to mutagenicity testing, Chem. Res. Toxicol., 4, 566-572. Davies, R.L., C.L. Crespi, K. Rudo, T.R. Turner and R. Langenbach (1989) Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens, Carcinogenesis, 10, 885-891. Djurid, Z., E.K. Fifer, Y. Yamazoe and F.A. Beland (1988) DNA binding by 1-nitropyrene and 1,6-dinitropyrene in vitro and in vivo: effects of nitroreductase induction, Carcinogenesis 9, 357-364.

Eddy, E.P., P.C. Howard, G.D. McCoy and H.S. Rosenkranz (1987) Mutagenicity, unscheduled DNA synthesis, and metabolism of 1-nitropyrene in the human hepatoma cell line HepG2, Cancer Res., 47, 3163-3168. Eddy, E.P., E.C. McCoy, H.S. Rosenkranz and R. Mermelstein (1986) Dichotomy in the mutagenicity and genotoxicity of nitropyrenes: Apparent effect of the number of electrons involved in nitroreduction, Mutation Res., 161, 109-111. Fifer, E.K., R.H. Heflich, Z. Djurid, P.C. Howard and F.A. Beland (1986) Synthesis and mutagenicity of 1-nitro-6nitrosopyrene and 1-nitro-8-nitrosopyrene, potential intermediates in the metabolic activation of 1,6- and 1,8-dinitropyrene, Carcinogenesis, 7, 65-70. Fu, P.P., M.W. Chou, D.W. Miller, G.L. White, R.H. Heflich and F.A. Beland (1985) The orientation of the nitro substituent predicts the direct-acting bacterial mutagenicity of nitrated polycyclic aromatic hydrocarbons, Mutation Res., 143, 173-181. Furth, E.E., W.G. Thilly, B.W. Penman, H.L. Liber and W.M. Rand (1981) Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates, Anal. Biochem., 110, 1-8. Gibson, T.L. (1986) Sources of nitroaromatic mutagens in atmospheric polycyclic organic matter, J. Air Pollution Control Assn., 36, 1022-1025. Hirayama, T., T. Watanabe, M. Akita, S. Shimomura, Y. Fujioka, S. Ozasa and S. Fukui, (1988) Relationships between structure of nitrated arenes and their mutagenicity in Salmonella typhimurium; 2- and 2,7-nitro substituted fluorene, phenanthrene and pyrene, Mutation Res., 209, 67-74. Howard, P.C., T. Aoyama, S.L. Bauer, H.V. Gelboin and F.J. Gonzalez (1990) The metabolism of 1-nitropyrene by human cytochromes P450, Carcinogenesis, 11, 1539-1542. Howard, P.C., J.A. Gerrard, G.E. Milo, P.P. Fu, F.A. Beland and F.F. Kadlubar (1983) Transformation of normal human skin fibroblasts by 1-nitropyrene and 6-nitrobenzo[a]pyrene, Carcinogenesis, 4, 353-355. Howard, P.C., K.A. Reed and D.R. Koop (1988) Oxidative metabolism of 1-nitropyrene by rabbit liver microsomes and purified microsomal cytochrome P-450 isozymes, Cancer Res., 48, 4261-4265. Imaida, K., M. Hirose, L. Tay, M.-S. Lee, C.Y. Wang and C.M. King (1991a) Comparative carcinogenicities of 1-, 2-, and 4-nitropyrene and structurally related compounds in the female CD rat, Cancer Res., 51, 2902-2907. Imaida, K., M.-S. Lee, C.Y. Wang and C.M. King (1991b) Carcinogenicity of dinitropyrenes in the weanling female CD rat, Carcinogenesis, 12, 1187-1191. International Agency for Research on Cancer (1983) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 32, Polynuclear Aromatic Compounds, Part 1, Chemical, Environmental and Experimental Data, IARC, Lyon. International Agency for Research on Cancer (1989) IARC Monographs on the Evaluation of Carcinogenic Risks to

W..F. Busby et aL / Mutation Research 322 (1994) 233-242 Humans, Vol. 46, Diesel and Gasoline Engine Exhausts and Some Nitroarenes, IARC, Lyon. Kinouchi, T., K. Nishifuji, H. Tsutsui, S.L. Hoare and Y. Ohnishi (1988) Mutagenicity and nitropyrene concentration of indoor air particulates exhausted from a kerosene heater, Jpn. J. Cancer Res. (Gann), 79, 32-41. Korfmacher, W.A., L.G. Rushing, J. Arey, B. Zielinska and J.N. Pitts Jr. (1987) Identification of mononitropyrenes and mononitrofluoranthenes in air particulate matter via fused silica gas chromatography combined with negative ion atmospheric pressure ionization mass spectrometry, J. High Resolution Chromatog. & Chromatog. Commun., 10, 641-646. Li, A.P. and J.S. Dutcher (1983) Mutagenicity of mono-, diand tri-nitropyrenes in Chinese hamster ovary cells, Mutation Res. Lett., 119, 387-392. Moiler, M.E. and S.S. Thorgeirsson (1985) DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells, Mutation Res., 151, 137146. Mori, H., S. Sugie, N. Yoshimi, T. Kinouchi and Y. Ohnishi (1987) Genotoxicity of a variety of nitroarenes and other nitro compounds in DNA-repair tests with rat and mouse hepatocytes, Mutation Res., 190, 159-167. Nachtman J.P. and S. Wolff (1982) Activity of nitro-polynuclear aromatic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation, Environ. Mutagenesis, 4, 1-5. Nakayasu, M., H. Sakamoto, K. Wakabayashi, M. Terada, T. Sugimura and H.S. Rosenkranz (1982) Potent mutagenic activity of nitropyrenes on Chinese hamster lung cells with diphtheria toxin resistance as a selective marker, Carcinogenesis, 3, 917-922. Otofuji, T., K. Horikawa, T. Maeda, N. Sano, K. Izumi, H. Otsuka and H. Tokiwa (1987) Tumorigenicity test of 1,3and 1,8-dinitropyrene in BALB/c mice, J. Natl. Cancer Inst., 79, 185-188. Paputa-Peck, M.C., R.S. Marano, D. Schuetzle, T.L. Riley, C.V. Hampton, T.J. Prater, L.M. Skewes, T.E. Jensen, P.H. Ruehle, L.C. Bosch and W.P. Duncan (1983) Determination of nitrated polynuclear aromatic hydrocarbons in particulate extracts by capillary column gas chromatography with nitrogen selective detection, Anal. Chem., 55, 1946-1954. Patton, J.D., V.M. Maher and J.J. McCormick (1986) Cytotoxic and mutagenic effects of 1-nitropyrene and 1-nitrosopyrene in diploid human fibroblasts, Carcinogenesis, 7, 89-93. Penman, B.W. and C.L. Crespi (1987) Analysis of human lymphoblast mutation assays by using historical negative control data bases, Environ. Molec. Mutagenesis, 10, 3560. Pitts, J.N., Jr. (1987) Nitration of gaseous polycyclic aromatic hydrocarbons in simulated and ambient urban atmospheres: a source of mutagenic nitroarenes, Atmos. Environ., 21, 2531-2547. Pitts, J.N., Jr., K.A. Van Cauwenberghe, D. Grosjean, J.P.

241

Schmid, D.R. Fitz, W.L. Belser Jr., G.B. Knudson and P.M. Hynds (1978) Atmospheric reactions of polycyclic aromatic hydrocarbons: facile formation of mutagenic nitro derivatives, Science, 202, 515-519. Ramdahl, T., B. Zielinska, J. Arey, R. Atkinson, A.M. Winer and J.N. Pitts Jr. (1986) Ubiquitous occurrence of 2nitrofluoranthene and 2-nitropyrene in air, Nature, 321, 425-427. Roscher E. and F.J. Wiebel (1992) Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines, Mutation Res., 278, 11-17. Rosenkranz, H.S. and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267. Rosenkranz, H.S. and R. Mermelstein (1985) The genotoxicity, metabolism and carcinogenicity of nitrated polycyclic aromatic hydrocarbons, J. Environ. Sci. Health, C3, 221272. Roy, A.K., K. El-Bayoumy and S.S. Hecht (1989) 32p-postlabeling analysis of 1-nitropyrene - DNA adducts in female Sprague-Dawley rats, Carcinogenesis, 10, 195-198. Saito, K., T. Kamataki and R. Kato (1984) Participation of cytochrome P-450 in reductive metabolism of 1-nitropyrene by rat liver microsomes, Cancer Res., 44, 31693173. Sawada, M., T. Sofuni and M. Ishidate Jr. (1991) Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity, Mutation Res., 264, 37-41. Shimada, T. and F.P. Guengerich (1990) Inactivation of 1,3-, 1,6-, and 1,8-dinitropyrene by cytochrome P-450 enzymes in human and rat liver microsomes, Cancer Res., 50, 2036-2043. Smith, B.A., W.A. Korfmacher and F.A. Beland (1990) DNA adduct formation in target tissues of Sprague-Dawley rats, CD-1 mice and A / J mice following tumorigenic doses of 1-nitropyrene, Carcinogenesis, 11, 1705-1710. Takayama, S., M. Tanaka, Y. Katoh, M. Terada and T. Sugimura (1983) Mutagenicity of nitropyrenes in Chinese hamster V79 cells, Jpn. J. Cancer Res. (Gann), 74, 338-341. Thornton-Manning, J.R., B.A. Smith, F.A. Beland and R.H. Heflich (1991) S9-Mediated metabolism of 1-nitropyrene to a mutagen in Chinese hamster ovary cells by ring-oxidation under aerobic conditions and by nitroreduction under anaerobic conditions, Carcinogenesis, 12, 2317-2323. Tokiwa, H. and Y. Ohnishi (1986) Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, Crit. Rev. Toxicol., 17, 23-60. Tokiwa, H., S. Kitamori, R. Nakagawa, K. Horikawa and L. Matamala (1983) Demonstration of a powerful mutagenic dinitropyrene in airborne particulate matter, Mutation Res., 121, 107-116. Tokiwa, H., R. Nakagawa and K. Horikawa (1985) Mutagenic/carcinogenic agents in indoor pollutants; the dinitropyrenes generated by kerosene heaters and fuel gas and liquefied petroleum gas burners, Mutation Res., 157, 3947.

242

W.F. Busby et al. /Mutation Research 322 (1994) 233-242

Wislocki, P.G,, E.S. Bagan, A.Y.H. Lu, K.L. Dooley, P.P. Fu, H. Han-Hsu, F.A. Beland and F.F. Kadlubar (1986) Tumorigenicity of nitrated derivatives of pyrene, benzo[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay, Carcinogenesis, 7, 1317-1322.

Yu, S., D. Herreno-Saenz, D.W. Miller, F.F. Kadlubar and P.P. Fu, (1992) Mutagenicity of nitro-polycyclic aromatic hydrocarbons with the nitro substituent situated at the longest molecular axis, Mutation Res., 283, 45-52.