Comparison of the mutant frequencies and mutation spectra of three non-genotoxic carcinogens, oxazepam, phenobarbital, and Wyeth 14,643, at the lambdacII locus in Big Blue transgenic mice.

Oxazepam (OX), a widely used benzodiazepine anxiolytic, phenobarbital (PHE), a drug used for convulsive disorders, and Wyeth 14,643 (WY; [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a hypolipidemic agent, are all hepatocarcinogenic in B6C3F1 mice. They have been classified as "non-genotoxic" carcinogens since they are non-DNA reactive in in vitro assays and are either negative or weakly positive in Salmonella typhimurium (Ames assay). Male B6C3F1 Big Blue(R) transgenic mice were fed 2500 ppm of OX or PHE or 500 ppm of WY in their diet, while a control group of mice received diet alone for 180 days. The mutant frequency (MF) of cII in the control mice, after correction for clonality, was 6.2 +/- 2.8 x 10(-5). The MF values for mice fed OX, PHE, and WY were 10.0 +/- 3.6 x 10(-5) (P < 0.05), 7.9 +/- 1.3 x 10(-5) (P = 0.1) and 17.4 +/- 4.2 x 10(-5) (P < 0.01), respectively. The mutation spectrum (MS) at cII from the PHE-fed mice was significantly different (P < 0.05) from that of the control mice even though the MF was not, whereas the MS spectra of mice fed OX (P = 0.4) and WY (P = 0.7) were not significantly different. The PHE-derived spectrum differed from the spontaneous spectrum in the lower occurrence of G:C>C:G transversions (17 vs 1.6%) and the higher incidence of A:T>T:A transversions (3.4 vs 9.5%). Prior to correction for clonal expansion, each treated group exhibited a high incidence of frameshift mutations at the homopolymeric run of guanines at bp 179-184 (OX 21%, PHE 21%, WY 16% of the total mutations); this was not the case with the control group (6%). Even after clonal correction, more than 10% of the mutations were frameshifts in the treated mice, while 5% were frameshifts in the control mice. Despite this hypersensitive region of the gene, our findings suggest that the cII locus is less sensitive than the lacI locus to mutation induction by non-DNA reactive carcinogens.

Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice.

The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations. Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed. Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment. We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice. A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups. While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not. The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007). The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI. The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C

LacI mutation spectra following benzo[a]pyrene treatment of Big Blue mice.

The mutation spectrum of the lacI gene from the liver of C57Bl6 Big Blue transgenic mice treated with benzo[a]pyrene (B[a]P) has been compared with the spectrum of spontaneous mutations observed in the liver of untreated Big Blue mice. Mice were treated with B[a]P for 3 days followed by a partial hepatectomy one day after the last injection. Liver tissue was removed for analysis at hepatectomy and, again, 3 days later at the time of sacrifice. Earlier, we reported that the lacI mutant frequency in these B[a]P-treated mice was elevated in the liver both at the time of hepatectomy and at sacrifice; however, a statistically significant increase in the mutant frequency was observed only at sacrifice. In this study, the DNA sequence spectra of lacI mutations observed in the liver of B[a]P-treated Big Blue mice at hepatectomy and at time of sacrifice were compared with each other and with the spectrum of spontaneous liver mutations. No differences were observed between the two B[a]P-treatment spectra. However, mutation frequencies of both GC-->TA and GC-->CG at the time of hepatectomy and at sacrifice were significantly elevated compared with the spontaneous frequency of these same transversions. Also, the frequency of AT-->TA transversions was significantly higher than the spontaneous frequency at the time of hepatectomy but not at sacrifice. The frequency of all other classes of mutations scored was not significantly different from the frequency of these same events in the spontaneous spectra. These data support the view that B[a]P treatment results in the induction of GC-->TA and GC-->CG transversions within 1 day of the last injection and they provide insights regarding the relative roles of benzo[a]pyrene-7,8-diol-9, 10-epoxide and radical cations of B[a]P in B[a]P-induced mutagenesis in vivo. Finally, these data provide evidence for B[a]P-induced mutagenesis under conditions where no statistical increase in mutant frequency could be shown.

Subchronic administration of phenobarbital alters the mutation spectrum of lacI in the livers of Big Blue transgenic mice.

Phenobarbital (PHE) is a liver carcinogen in B6C3F1 mice and a weak mutagen that does not appear to form DNA adducts. To investigate PHE mutagenicity in vivo, B6C3F1 Big Blue(R) male transgenic mice harboring the lambdaLIZ shuttle vector containing the lacI target gene were fed PHE at 2500 ppm for 180 days. A modest increase in the mutant frequency (MF) from 5.02+/-2.4x10(-5) in the control group to 6.88+/-0.754x10(-5) in the PHE-treated group, which was marginally different (p<0.05), was obtained. To better assess the relevance of this increase in MF, a random collection of mutants from each PHE-exposed mouse was sequenced. After correcting for clonal expansion, which is the most conservative approach, the MF in the PHE-treated mice decreased to 6.39+/-1.02x10(-5), an insignificant difference (p=0.10) from that in control group. Despite this modest increase in MF, the mutation spectrum obtained from the PHE-exposed group was significantly different (pA:T transitions remained the same in the two spectra. It is postulated that the increase in transversions at G:C base pairs found in the PHE-derived spectrum is likely due to oxidative damage as a result of induction of CYP2B isozymes by the chronic administration of PHE. Results from this study demonstrate that PHE alters the spectrum of mutations, rather than inducing a significant global increase in the MF. The PHE-derived spectrum of lacI mutants from the liver of Big Blue(R) B6C3F1 male mice was remarkably similar (p=0.8) to that generated by oxazepam (OX), a compound which also induces CYP2B isozymes following chronic administration of the drug.

Urinary mutagenicity as an indicator of occupational exposure in a cohort of cosmetologists.

Epidemiological studies suggest a higher risk of hematopoietic disorders including lymphoma among cosmetologists. The etiology of these disorders among cosmetologists is unknown, but beauticians are exposed to a wide variety of chemicals in the workplace. In this study, the urinary mutagenicity of cosmetologists was studied as an indicator of occupational exposure. A microsuspension modification of the Ames assay with Salmonella typhimurium strain TA98 was used to detect direct-acting mutagens and promutagens in urine. A comparable group of teachers of similar age and gender, and living in the same geographic area was used as the control group. There was no elevated risk for urinary mutagenicity among the cosmetologists after controlling for a number of confounders including smoking. In a multivariate model, smoking regularly or within 24 h of sample collection was found to be positively associated with urinary mutagenicity among both groups. The number of cigarettes smoked daily, age, and length of employment were not associated with urinary mutagenicity. Analysis of urine samples collected successively from each participant showed a fair to good agreement between promutagens in samples, suggesting a fairly constant exposure to promutagens.

Oxazepam is mutagenic in vivo in Big Blue transgenic mice.

Although oxazepam (Serax), a widely used benzodiazepine anxiolytic, does not induce gene mutations in vitro or chromosomal aberrations in vivo, it was found to be a hepatocarcinogen in a 2 year bioassay in B6C3F1 mice. Thus, it was of interest to determine whether this carcinogen is mutagenic in vivo. Male B6C3F1 Big Blue transgenic mice were fed 2500 p.p.m. oxazepam or control diet alone for 180 days and killed on the next day. The mutant frequency (MF) of lacI in control mice was 5.02 +/- 2.4x10(5), whereas the MF in the oxazepam-treated mice was 9.17 +/- 4.82x10(-5), a significant increase (P < 0.05). Correction of the mutant frequency of lacI from the oxazepam-treated mice for clonality resulted in a decrease in the mean mutant frequency to 8.15 +/- 2. 54x10(-5). Although the mutant frequency difference was small, sequencing of a random collection of the mutants from each oxazepam-exposed mouse showed a significant difference (P < 0.015) in the mutation spectrum compared with that from control mice. In the oxazepam-exposed mice, an increase in G:C-->T:A and G:C-->C:G transversions and a concomitant decrease in G:C-->A:T transitions were observed. Clonal expansion of mutations at guanines in 5'-CpG-3' sequencing contexts at three sites was noted. It is postulated that some of the mutations found in the oxazepam-derived spectrum were due to oxidative damage elicited by induction of CYP2B isozymes as the result of chronic oxazepam administration. This study demonstrates that the in vivo Big Blue transgenic rodent mutation assay can detect mutations derived from a carcinogen that did not induce gene mutations in vitro or micronuclei in mouse bone marrow. Moreover, the sequencing of the recovered mutants can distinguish between the mutation spectrum from treated mice compared with that from control mice, thereby confirming the genotoxic consequences.

Mutant frequency of lacI in transgenic mice following benzo[a]pyrene treatment and partial hepatectomy.

The Big Blue, transgenic mouse provides an in vivo mutation system that permits the study of pharmacodynamic parameters on mutant frequency (MF) following xenobiotic exposure. We have studied the effects of cellular proliferation on the frequency of mutations in the lacl transgene by evaluating the MF in the liver of male C57B1/6 Big Blue mice following treatment with benzo[a]pyrene (B[a]P) and a partial hepatectomy. Mice received either 40 mg/kg of B[a]P in corn oil or corn oil alone by i.p. injection on three consecutive days, followed by a partial hepatectomy on the fourth day. Three days later (i.e., 7 days following the initial B[a]P injection), the animals were sacrificed and the MF in the liver was compared to the MF observed in the liver of the same mouse at the time of hepatectomy. Induction of cytochrome P-450 1A (CYP1A) following B[a]P treatment was evident by Western blot analysis. The MF in untreated control animals was not significantly different at hepatectomy (4.7 +/- 0.8 x 10(-5)) and 3 days later, at sacrifice (3.0 +/- 0.4 x 10(-5)). Neither was the MF observed in the B[a]P-treated mice at the time of sacrifice (12.0 +/- 2.1 x 10(-5)) significantly different from the MF observed at the time of hepatectomy (10.6 +/- 5.3 x 10(-5)). However, B[a]P-treatment resulted in a 4.0-fold increase in MF at sacrifice which was significantly different (p < 0.05), when compared to the untreated controls. The B[a]P-treated mice at hepatectomy showed a modest 2.2-fold increase in MF which was not statistically significantly different from the untreated controls. In addition, both control and B[a]P-treated tissues gave sectored mutant plaques. The sectored plaque frequency (SPF) was significantly elevated (p < 0.05) in the B[a]P-treated mice at hepatectomy (4.2 +/- 1.0 x 10(-5)) and sacrifice (7.3 +/- 2.4 x 10(-5)) as compared to the respective frequency in the control mice at hepatectomy (1.9 +/- 0.7 x 10(-5)) and sacrifice (1.4 +/- 0.2 x 10(-5)). One explanation for this data is the persistence of the B[a]P adducts in the mouse genomic DNA that was packaged into the lambda phage, and ultimately fixed as mutations in Escherichia coli.

Activation and detoxification of dinitropyrenes by cytosol and microsomes from Aroclor-pretreated rats in the Ames and umu assays.

1,3-, 1,6-, and 1,8-Dinitropyrene (1,3-, 1,6-, and 1,8-DNP) are direct-acting mutagens in that they do not require an exogenous source of enzymes for activation to mutagens in the Ames assay. However, the addition of mammalian S9 preparations, or the microsomal and cytosolic compartments comprising S9, modulate the mutagenic response of these DNPs. In this study, we compared the mutagenic response of these DNPs in the presence of cytosol and microsomal fractions from the liver of Aroclor-pretreated (AR) and control rats, in the Ames mutagenicity assay and umu gene induction assay. 1,3- and 1,8-DNP were deactivated to a greater extent by microsomes from AR-induced and control rats than was 1,6-DNP, in both the umu and Ames assays. In the Ames assay, S9 was more potent in deactivating the DNP than an equivalent concentration of microsomes from the same S9 preparation. Also, S9 from AR-pretreated rats deactivated the isomers to a greater extent than S9 from control rats. In contrast to the constant deactivation of all the isomers in the two assays catalyzed by microsomes and S9, the response with cytosol from AR-pretreated rats differed with respect to the three isomers in the Ames and umu assays. When cytosol from AR-treated rats was added, the mutagenicity of 1,3- and 1,6-DNP, but not 1,8-DNP, was significantly (P < 0.05) increased in the Ames assay while the mutagenicity of the three DNPs was increased in the umu assay. Also, a biphasic response was observed in the umu assay with 1,6- and 1,8-DNP, in that AR-cytosol enhanced the mutagenicity at low protein concentrations (5-50 micrograms protein/reaction) but abrogated the response at higher protein concentrations. The effect of cytosol from control rats depended on the isomer tested; 1,3-DNP was activated above the background level in both assays (nearly towfold) while 1,6-DNP and 1,8-DNP were only activated at low protein concentrations in the umu assay. In the Ames assay, cytosol from AR-pretreated rats did not alter the mutagenic response with 1,8-DNP, while control cytosol significantly (P < 0.05) deactivated 1,8-DNP at all substrate concentrations tested. In summary, this study showed that the mutagenicity of 1,3-DNP was similar in the two assays but the responses with 1,6- and 1,8-DNP differed in the two assays. These isomeric differences could be due to the varying metabolic pathways of the three DNPs as well as the detectable end points of the two assays.

Regioselective hydroxylations of 2-nitrofluorene in vivo: a nuclear magnetic resonance study.

The time-dependent metabolism of 2-nitrofluorene (2-NF) in vivo has been studied after ip administration to rats. After hydrolysis with beta-glucuronidase/arylsulfatase, five hydroxylated 2-nitrofluorenes were isolated by HPLC and identified by a combination of data from NMR, UV, and MS experiments. These metabolites were characterized as 6,9-dihydroxy-2-nitrofluorene (I), 9-hydroxy-2-nitrofluorene (II), 6-hydroxy-2-nitrofluorene (III), 7-hydroxy-2-nitrofluorene (IV), and 8-hydroxy-2-nitrofluorene (V). The highest amounts of metabolites were found after 24 h of injection, and although the major ones were still present after 48 h, only traces of them could be found beyond that time. A unique feature of the present study was our ability to establish the position of the different hydroxylations that occur in vivo by NMR techniques and, thus, to observe the regioselectivity of the metabolism of the 2-NF in vivo. Furthermore, two conjugated metabolites were identified by 1H-NMR and ESI-MS as 6-[(hydroxysulfonyl)oxy]-2-nitrofluorene (2-nitrofluorene 6-sulfate) (VI) and 7-[(hydroxysulfonyl)oxy]-2-nitrofluorene (2-nitrofluorene 7-sulfate) (VII).

Distinction of mutagenic carcinogens from a mutagenic noncarcinogen in the big blue transgenic mouse.

The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics. Both compounds are rapidly absorbed after oral administration and are equally mutagenic in the Ames test; however, 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses. The Big Blue transgenic B6C3F1 mouse carries multiple copies of the lacl mutational target gene. Our studies were designed to determine whether the Big Blue system could be used to detect differences in the vivo mutagenic activity between the carcinogen-noncarcinogen pair 2,4-DAT and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results. Male B6C3F1 transgenic mice were exposed to 2,4-DAT or 2,6-DAT at 0 or 1,000 ppm in the diet for 30 and 90 days or to dimethylnitrosamine as a positive control. Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (p < 0.01) as compared to both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)). Results from this study demonstrate that the Big Blue transgenic mutation assay can distinguish differences in vivo between the mutagenic responses of hepatic carcinogens ad a noncarcinogen; is sensitive to mutagens through subchronic dietary exposure; and yields a differential response depending upon the length of time mice are exposed to a mutagen.

Differential in vivo mutagenicity of the carcinogen/non-carcinogen pair 2,4- and 2,6-diaminotoluene.

The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics. Both compounds are equally mutagenic in the Ames/Salmonella assay in the presence of S9. However, the differences in the results of chronic rodent carcinogen bioassays using these two compounds are significant, in that 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses. The Big Blue transgenic B6C3F1 mouse carries multiple copies of bacteriophage lambda, each with a lacI mutational target gene, integrated into mouse chromosome 4. Our studies were designed to determine whether the Big Blue system could be used to detect differences in the in vivo mutagenic activity between the carcinogen/non-carcinogen pair 2,4- and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results. Male B6C3F1 transgenic mice were exposed to 2,4- or 2,6-DAT at 0 or 1000 p.p.m. in the diet for 30 and 90 days. Mice serving as positive controls were administered five daily i.p. injections of 6 mg/kg dimethylnitrosamine (DMN) in saline and were sacrificed 15 days following the last injection. Mutant frequencies at lacI were determined by recovering the genomically integrated lambda phage using an in vitro packaging reaction followed by infection of an appropriate Escherichia coli host. Complete non-sectored blue mutant plaques were scored against a background of clear non-mutant plaques. Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (P < 0.01) as compared with both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)). Mutations at lacI arising ex vivo during replication in E. coli are observed in this system as sectored blue plaques. The sectored plaque frequency in this study was constant across all groups at approximately 9.0 x 10(-5). Results from this study demonstrate that the Big Blue transgenic mutation assay: (i) can distinguish differences in vivo between the mutagenic responses of a carcinogen and a non-carcinogen which elicited comparable mutagenic activity in S.typhimurium; (ii) is sensitive to mutagens through subchronic dietary exposure; and (iii) yields a differential response depending upon the length of time mice are exposed to a mutagen.

Variability over time in the mutagenicity of ashes from municipal solid-waste incinerators.

Incineration of municipal solid waste as an alternative to its disposal in landfills has advantages such as volume reduction and generation of energy. However, both air emissions and the residual ash may pose environmental and human health hazards. The Ames mutagenicity assay was used to determine the mutagenicity of fly and bottom ash from two incinerators over time. This assay is an alternative to costly and time-consuming chemical analyses and is more realistic for the assessment of the best disposition of the ash i.e. whether it could pose a risk to handlers of the ash, whether it can be used in cement or as a fertilizer or whether it should be relegated to a landfill. The mutagenic potency of fly and bottom ash on a per g weight basis of material is similar. Furthermore, the variability over time in mutagenicity indicates that constant monitoring of incineration products and byproducts is essential.

Modulation of the mutagenicity of three dinitropyrene isomers in vitro by rat-liver S9, cytosolic, and microsomal fractions following chronic ethanol ingestion.

The effects of chronic ethanol feeding of rats on the ability of liver fractions to modulate the bacterial mutagenicity of three dinitropyrene isomers (1,3-, 1,6- and 1,8-DNP), which require bacterial enzymes but not an exogenous enzyme source for activation, were studied. The mutagenicity of the DNP isomers toward S. typhimurium TA98 and TA100 was attenuated in the presence of post-mitochondrial supernatants (S9) from both ethanol-fed and pair-fed rats albeit, that from the ethanol-fed group was more efficient in lowering the mutagenicity. The cytosolic fraction from ethanol-fed rats enhanced the mutagenicity of all of the DNP isomers in TA100. The most notable enhancement was with 1,3-DNP in which a more than 4-fold enhancement was obtained. Cytosol from pair-fed rats enhanced only the mutagenicity of 1,3-DNP, this by 2.9-fold. Cytosolic NADPH-nitroreductase activity from ethanol-treated rats toward 1,6-, 1,8- and 1,3-DNP was increased 2.8-, 1.7- and 1.3-fold, respectively over pair-fed controls. Cytosolic NADH-nitroreductase from ethanol-fed rats was increased with 1,3-DNP (1.7-fold) and 1,8-DNP (1.4-fold) as substrates, but not with 1,6-DNP. Microsomes decreased the mutagenicity of DNP similarly to S9, i.e., fractions from ethanol-fed rats were more efficient than those of pair-fed rats in deactivating all the DNP isomers. Per mg of protein, detoxification of DNP by S9 was more efficient than with microsomes, thus both cytosolic and microsomal enzymes are required for maximal detoxification. In summary, ethanol feeding modulates both the augmented cytosolic activation of DNP to mutagens and the deactivation of the direct-acting mutagenicity of DNP by microsomes. In combination, as is the case with S9, the microsomal detoxifying activity outcompetes the cytosolic activation.

Arylamine activation following chronic ethanol ingestion by rats: studies on the liver S9, microsomal and cytosolic fractions and comparison with Aroclor 1254 pretreatment.

That enzyme fractions derived from animals chronically fed alcohol can alter the metabolism of carcinogenic xenobiotic compounds has been documented. To further understand this relationship the mutagenicity of 3 aromatic amines was determined in the Ames test, employing activation systems derived from rats maintained on an alcohol-containing liquid diet, an isocaloric control liquid diet or Aroclor 1254-pretreated animals fed standard laboratory chow. Depending upon protein and substrate concentrations, S9 from ethanol-fed rats was 30-50% less efficient than S9 from pair-fed rats in activating arylamines (2-aminofluorene, 2-aminoanthracene and 2-acetylaminofluorene) to mutagens in Salmonella typhimurium TA98 and TA100. Cytosolic fractions from ethanol-fed animals always resulted in greater arylamine activation than that of controls whereas the opposite was true of the microsomal compartment in which the ethanol-treated group was consistently less active than the controls. The cytosolic N-acetyltransferase activities with respect to 2 different substrates, isoniazid and 2-aminofluorene, were unaffected by ethanol consumption, indicating that this activity probably does not account for the different activation profiles exhibited by the ethanol and pair-fed cytosolic systems. Both the cytosolic and microsomal compartments are required for maximal expression of the mutagenicity of each arylamine however, each compartment can activate arylamines independently of the other. Reconstituting cytosol with microsomes from ethanol- and pair-fed rats, but not Aroclor-pretreated rats, resulted in a synergistic activation of the aromatic amines and displayed an effect similar to that of S9. Compared to Aroclor pretreatment and pair-fed controls, microsomes from ethanol-fed rats displayed the least capacity for activating any of the arylamines to mutagens. Microsomes from Aroclor-pretreated rats accounted for at least 80% of the S9-mediated activation of each of the arylamines to mutagenic metabolites which was in marked contrast to the contribution of the microsomal fractions to the S9 activity in the ethanol- (5-20% of S9 activity) and pair-fed systems (22-30% of S9 activity). The data indicate that 2 opposing reactions occur in S9, a cytosolic activity that augments and a microsomal activity that attenuates the mutagenicity of arylamines. Both activities are modified by ethanol consumption and Aroclor pretreatment.

Comparative mutagenicity of nitrofluoranthenes in Salmonella typhimurium TA98, TA98NR, and TA98/1,8DNP6.

The nitration of fluoranthene, one of the most abundant polycyclic aromatic hydrocarbons (PAH) in diesel fuels, occurs in the laboratory under either electrophilic or free-radical conditions to give nitro-PAH. 3-Nitrofluoranthene (3-NF) is the major product under electrophilic ionic conditions while 2-nitrofluoranthene (2-NF) is the major product under free-radical nitration conditions. The free-radical nitration of fluoranthene also yields 1,2- and 1,3-dinitrofluoranthene (1,2-DNF and 1,3-DNF). Nitration on the 3-position of fluoranthene enhances the mutagenic potency more strongly than on the 2-position. Thus, 3-NF is a more potent mutagen than 2-NF and 1,3-DNF is more potent than 1,2-DNF, an isomer with one near coplanar nitro group and one nitro group substantially out of plane with the fluoranthene skeleton, when tested against Salmonella typhimurium TA98, TA98NR, and TA98/1,8-DNP6. In addition, the activation of these dinitro-PAH to mutagens does not depend on the "classical nitroreductase" and/or O-acetylase, suggesting that they are activated via different pathways. Despite the fact that 3-NF and 1-phenyl-4-nitronaphthalene (1-P-4NN), a non-planar analog of 3-NF, have virtually identical reduction potentials, their mutagenic potencies differ by three orders of magnitude. This finding suggests that when nitro-PAH of varying steric requirements are compared, the reduction potential may not predict mutagenic potency as well as had been previously suggested.

1H NMR studies on mutagenic nitrofluoranthenes and exposure risk assessment.

The 1H NMR spectra of the nitrofluoranthene isomers are presented to allow gas chromatographic analysis of environmental samples suspected of containing nitrofluoranthenes. The mutagenic isomers 1-, 2-, 3-, 7-, and 8-nitrofluoranthene and 1,2- and 1,3-dinitrofluoranthene and a nonmutagenic analogue, 1-phenyl-4-nitronaphthalene, have been studied by using nuclear Overhauser effects and 2D shift-correlated 1H NMR spectroscopy. The X-ray crystal structure of 1-phenyl-4-nitronaphthalene is also reported. Reduction potentials and coplanarity of the nitro group have been used to correlate the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitro-PAH) measured by the Ames assay, but our data suggest that these parameters are not sufficient for the prediction of mutagenic potency in the nitrofluoranthene series.

Organic toxicants and mutagens in ashes from eighteen municipal refuse incinerators.

Ashes, obtained from about one-fourth of the operating municipal refuse incinerators in the United States, were analyzed for a range of organic toxicants and mutagens. Thirty percent of the ash samples, which consisted of bottom ash or bottom ash-fly ash mixtures, contained 20-74% organic matter. Thirty percent of the ashes contained direct-acting and/or promutagens which revertedSalmonella typhimurium TA98 or TA100. Sixty percent of the ashes contained more than 5 ng/g of polychlorinated biphenyls. The concentration of tetra- and pentachlorinated biphenyls were higher than the mono-, di-, hepta- and octachlorinated biphenyls. A similar distribution of congeners was seen in polychlorinated dibenzodioxins found in the ashes. The major volatileN-nitroso compounds found in the ashes wereN-nitrosodimethylamine andN-nitrosomorpholine. Other classes of compounds which were found in the ashes included chlorinated benzenes, phthalates, and substituted benzothiophenes.

Effects of inhaled municipal refuse incinerator fly ash in the guinea pig.

Fly ash was collected from two municipal refuse incinerators. It was analyzed for heavy metals, elements, and a wide range of toxic organics. It was resuspended in air for inhalation exposure of guinea pigs. These animals were exposed at high concentrations of each ash 6 h/d for 5 d, and tissues were taken 45 d after the exposure. Following the first exposure and after each daily exposure the ventilatory response of these animals upon challenge with CO2 was found to be depressed. Recovery occurred following exposure. Heavy metals, cadmium, lead, zinc, and mercury were elevated in the lungs of these animals. Histologic evaluation of pulmonary tissue revealed multifocal pneumoconiosis. Interstitial infiltration by macrophages and smooth muscle hypertrophy of blood vessels and bronchioles were also observed. There was no evidence of a dioxinlike toxic effect following inhalation of these ashes.

Activation of two environmental mixtures by plant S9.

Nitrated and ozonized pyrene mixtures were assayed for their mutagenic activity in the presence or absence of pea S9 using Salmonella typhimurium TA98 as the indicator organism. The plant enzymes increased the mutagenic response of these mixtures above that obtained in the absence of S9. The optimum S9 protein concentration for the activation of the nitrated pyrene mixture at 0.1 microgram was 3.9 mg/plate whereas that for the ozonized pyrene mixture at 33.3 micrograms was 3.2 mg protein/plate. BSA could not replace S9, and NADPH was a required co-factor in the activation of both mixtures by pea S9. Although the nitrated pyrene mixture was determined to consist of approximately 90% 1-nitropyrene, the mutagenic response due to this compound ranged from 30 to 50% of that of the mixture.