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Wastewater collected from oil-water separating tanks of ten gasoline stations for a year was fractionated into diethyl ether-soluble neutral, acidic, and basic fractions. Mutagenicity of these fractions was measured with Salmonella typhimurium strains TA98 and TA100 in the presence or absence of S9 mix. The neutral fractions showed high mutagenicity in the absence of S9 mix. Each neutral fraction was subjected to high-performance liquid chromatography (HPLC) and fractionated. A 1-nitropyrene(1-NP)-corresponding fraction was collected and analyzed by gas chromatography-mass spectrometry (GC-MS) and HPLC to prove that wastewater contains 1-NP and to quantitate 1-NP in wastewater. GC-MS patterns showed the following molecular and fragment ion peaks of 1-NP: 247, 217, 201, and 189. The amount of 1-NP in 36 samples of wastewater was 4.2-25,600 ng per liter of wastewater, and 1-NP accounted for 0.3-58.5% of the total mutagenicity of the neutral fractions. The other 19 samples of wastewater did not contain any detectable 1-NP. The mutagenicity of wastewater may be due to water from car washing and contamination by used crankcase oil. A Soxhlet extract of crankcase oil used in a gasoline was fractionated into three fractions as above. Mutagenicity was measured with strains TA98, TA100, TA98NR, and TA98/1,8-DNP6 in the absence or presence of S9 mix. The neutral fraction showed the highest mutagenicity with strain TA98 in the absence of S9 mix, and its mutagenicity was decreased in strains TA98NR and TA98/1,8-DNP6. The latter result indicates that the used engine-oil contained 1-NP and dinitropyrenes. Actually, the amounts of 1-NP and 1,6-diNP in used crankcase oil were 138 and 2.0 ng per ml of oil, respectively, and these concentrations accounted for 0.45 and 2.7%, respectively, of the total mutagenicity of the neutral fraction with strain TA98 in the absence of S9 mix. Moreover, the concentrations of 1-NP and 1,6-diNP in used crankcase oil of a diesel engine were 349 and 31 ng per ml of oil, respectively, accounting for 0.9 and 12%, respectively, of the total mutagenicity of the neutral fraction in the same assay system.

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.

A mutant that rapidly degrades more than 80% of its rRNA and tRNA under defined conditions was genetically analyzed. Two genes, srnA and srnB, are separately located, and the mutated alleles of both are required for degradation of stable RNA in cultures treated with rifampicin at 42°. srnA is closely linked to tsx by matings and transduction tests; by P1 transduction, the gene order is lac (9 min) proC (9.55 min) tsx (9.8 min) srnA (about 10 min) purE (12 min) rnsA (14.4 min). srnB is not yet completely mapped, but is outside the lac-rnsA region, probably in the region between 75 and 90 min.—The product of the rnsA gene, RNase I, is a potent endonuclease of E. coli, and the only one known that can attack ribosomes and tRNA. However, not only are the srn lesions genetically separate from rnsA, but also, derivatives of an srn strain were prepared lacking RNase I, and they retain the Srn- phenotype. Thus, no correlation of rapid RNA turnover and RNase I activity has been found.

To investigate the modifying role of the intestinal microflora in the metabolism of 1-nitropyrene (1-NP) via enterohepatic circulation, we collected bile from male Wistar rats administered [3H]1-NP orally. The bile was mixed with the intestinal contents (IC) prepared from untreated rats and the mixture was incubated anaerobically under an atmosphere of nitrogen at 37 C. Samples of the reaction mixture were removed at intervals to assay their mutagenic potential, to determine the radioactivity bound to the IC, and for analysis of the biliary metabolites. The binding of the radioactivity to the IC increased linearly as a function of time during the 1-hr incubation. The time-dependent binding does not occur with heat-treated IC and the binding was inhibited by addition of D-saccharic acid 1,4-lacton, a beta-glucuronidase inhibitor. The mutagenicity (for Salmonella typhimurium strain TA98 without S9 mix) of the bile increased early in the incubation period and then decreased very rapidly. The mutagenicity of the bile was also enhanced by treatment with a sonicated IC extract or beta-glucuronidase, but not with a heat-treated IC or aryl-sulfatase. The metabolites produced after the bile was incubated for short periods with the IC were mainly nitrohydroxypyrenes; at later times nitroreduction occurred. The level of acetylaminohydroxypyrenes, which were formed by deconjugation, did not change during the incubation. To determine the degree of contribution of the IC to the total acetylating capacity, we measured acetyltransferase activity of the IC and various organs in Wistar rats. The liver had the highest N-acetyltransferase activity among the seventeen organs examined. Considerable activity was also detected in the kidney, small intestine, lung, and testis, but the IC showed very low activity. The acetylating capacity of the IC was 0.27% of the total capacity in rats, and that of the liver was more than 80%. These results suggest that the nitrohydroxypyrenes formed from 1-NP in the liver were conjugated to glucuronic acid and excreted via the bile duct into intestine. Hydrolysis of these glucuronide conjugates by bacterial beta-glucuronidase liberated into intestine, free nitrohydroxypyrenes, which were direct-acting mutagens. The released aglycons were then rapidly nitro-reduced by intestinal microflora, but contribution of the intestinal microflora to acetylation of the reduced metabolites is very low.

The particulates in a room warmed with a radiant kerosene heater were collected, extracted and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA98, TA98NR, TA98/1,8-DNP6 and TA100 in the presence and ahsence of S9 mix. Room air without the heater showed very low mutagenicity. However, a sample from a room at the beginning of the burning period showed very high mutagenicity (237 His+ revertants/plate/m*3 of air in strain TA98 in the absence of S9 mix). In contrast, emissions from the heater after it was burning stably showed low mutagenicity (9 His+ revertants/plate/m*3). The crude extract of particulates from the heater at the beginning of the burning period was analyzed by high-pressure liquid chromatography (HPLC) and showed a considerable amount of nitropyrenes (NPs); the concentrations of 1-NP and 1,6-diNP were 1.62 ng and 0.149 ng/m*3 of air, respectively, and accounted for 1.2% and 17.6%, respectively, of the mutagenicity in strain TA98 in the absence of S9 mix. In addition, an HPLC-Ames histogram showed that peaks of mutagenicity corresponding to 1-NP and diNPs accounted for 75.7% (1-NP, 4.9%; 1,6-diNP, 17.1%; 1,8-diNP, 46.3%; 1,3-diNP, 7.4%) of the HPLC-recovered mutagenicity for strain TA98 without S9 mix. These results suggest that kerosene heaters, especially immediately after ignition, create mutagenic substances such as NPs.

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.

Nitroarenes are postulated to play a principal part among mutagens/carcinogens which are induced in the combustion process and, in addition, are widely distributed in the environment. This review deals with the following points concerning nitroarene toxicity. Data on the mutagenicity of nitroarenes obtained by short-term bioassays are expected to provide us with sufficient information for us to determine their genotoxicity and carcinogenicity. Therefore, mutagenicity detected with Salmonella, Escherichia, and yeast test systems is discussed. Genotoxicity in mammalian cells is also important for determining the mutagenic properties of nitroarenes. In this article, mutagenicity in Chinese hamster ovary cells, sister chromatid exchanges, and cell transformation is summarized. The metabolism of nitroarenes in vivo and in vitro is of importance for determining their behavior and active forms. Therefore, current studies regarding metabolism of nitroarenes are described. Carcinogenicity of nitroarenes for animals has been reported by many workers. In this review, the incidence and histological features of tumors induced by nitroarenes are described. Furthermore, the possible association between human lung cancer and nitroarenes is discussed. Sources of nitroarenes in the environment are given. The results of various chemical tests for identifying nitroarenes are summarized, and speculation on the risk of nitroarenes for humans is presented.

Pieces of raw chicken with or without a marinating sauce were grilled over a city gas flame, extracted with benzene-ethanol (4:1) by ultrasonication and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor 1254-treated Sprague-Dawley rat liver (S9 mix). The basic fraction of yakitori without the sauce was more mutagenic than the other fractions for S. typhimurium strain TA98 in the presence of S9 mix. This is probably due to the presence of amino acid or protein pyrolysates. However, when the chicken was grilled with the sauce, the basic fraction showed lower mutagenicity for strain TA98 in the presence of S9 mix than did the same fraction without the sauce. The neutral fraction of yakitori with sauce showed high mutagenicity for strain TA98 in the absence of S9 mix, but low mutagenicity for strains TA98NR and TA98/1,8-DNP6, suggesting that this fraction might contain nitropyrenes (NPs). The neutral fraction of yakitori was analyzed by high-performance liquid chromatography (HPLC). The neutral fraction of the chicken grilled with the sauce for 3, 5 and 7 min contained 3.8, 19 and 43 ng, respectively, of 1-NP per gram of yakitori accounting for 3.0, 2.7 and 1.3%, respectively, of the total mutagenicity.(ABSTRACT TRUNCATED AT 250 WORDS)

An intracellular membrane-free, trypsinlike protease was isolated from cells of Bacteroides gingivalis 381. The protease was extracted from the cells by ultrasonic treatment and was purified about 250-fold with a recovery of 2% by sequential procedures. The properties of the protease were as follows: its optimal pH was 8.5; its activity was almost completely lost on incubation at 50 degrees C for 15 min; its activity was inhibited by diisopropylfluorophosphate, p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, Mn2+, Cu2+, and Zn2+; it hydrolyzed casein, azocasein, N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), bovine serum albumin, azocoll, and gelatin, but not N-alpha-benzoyl-DL-lysine-p-nitroanilide or human serum immunoglobulin A; its molecular weight was estimated as 45,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and its Km values for azocasein and BAPNA were 1.11% and 0.19 mM, respectively.

Using the Ames Salmonella-microsome system, we detected mutagenic activity in the exhaust from two kinds of 4-cycle gasoline engines of unregulated and regulated cars, and from diesel engines, as well as in the particulates from air collected in tunnels. The mutagenicity of particulates from a car equipped with a catalyst (regulated car), as compared with that from an unregulated car, was reduced very much (down to 500 from 4500 revertants/plate/m 3 in tester strain TA98). However, the mutagenicity of the ether-soluble acid and neutral fractions from the condensed water of emissions from a regulated car was still high (down to 2880 from 10 900 revertants/plate/m 3 in tester strain TA100). The mutagenic activity of emission exhaust from old diesel car engines was very high; the particulates showed 9140 and 19 600 revertants/plate/m 3 from strain TA98 incubated with an activating rat-liver S9 fraction. A small diesel engine of the type used for the generation of electric power or in farm machinery also produced exhaust with highly mutagenic particulates. The mutagenic activity of a methanol extract of particulate air pollutants collected in a highway tunnel showed 39 revertants/plate/m 3 toward strain TA98 and 87 toward strain TA100. The ether-soluble neutral fraction yielded 86 revertants/plate/m 3 from strain TA98 and 100 from strain TA100. This fraction also contained carcinogenic compounds, including benzo[ a ]pyrene, benzo[ e ]pyrene, benz[ a ]anthracene, benzo[ ghi ]perylene and chrysene. Very high mutagenic activity was detected, especially in the particulate air pollutants collected at night, in another tunnel on a superhighway: 60–88 revertants/plate/m 3 from strain TA100 for the sample collected by day, but 121–238, by night. Night traffic includes many more diesel-powered vehicles compared with gasoline-powered automobiles.

The srnB+ gene located on the F plasmid was assayed for its capacity to facilitate the release from infected cells of phage λ lacking the usual lytic activity. The srnB+ plasmid pOY54, carrying the 1.4–2.5F fragment in the EcoRI-BamHI fragment of pBR322, induced bacteriolysis and the release of progeny phage of the λcI857 susS7 lysogen in the presence of rifampin at 42°C. An srnB1 mutant plasmid, pOY541, did not promote bacteriolysis. These results suggest that the srnB+ gene of the F plasmid complements the function of the λ S gene in the nonpermissive host strain.

The pnd gene promotes the degradation of stable RNA in the presence of rifampicin at 42 degrees C, but is repressed during normal growth (Ohnishi, Y. and Akimoto, S. (1980) J. Bacteriol. 144, 833-835). We have determined the sequence of a third srnB-pnd-type gene, and have analyzed the effects of its expression from an inducible promoter. The nucleotide sequence of the pnd gene of the R16 plasmid exhibits an open reading frame for a polypeptide with 50 amino-acid residues, with high sequence homology to the pnd gene of a plasmid (R483) of a different incompatibility group. A possible base-paired stem and loop structure, which may participate in the regulation of gene expression, was detected between the promoter and the initiation codon, analogous to that in two comparable genes, srnB in the F and pnd in the R483 plasmid. When bacterial cells containing a lac-pnd fusion plasmid were incubated with a lac inducer at 30 degrees C, magnesium was released from the cells in bulk, and spheroplasts of the cells lysed even in hypertonic solution. Furthermore, when Mg2+ efflux was inhibited in the medium containing 5 mM Mg2+ or in Tris-HCl buffer, the degradation of stable RNA at 42 degrees C was inhibited. These results suggest that expression of the pnd gene effects a release of cellular magnesium by a membrane alterations, resulting in the stable RNA degradation at a higher temperature.

When 25 kinds of Japanese soy sauce at a concentration of 5% were incubated with 50mM sodium nitrite (pH 2) at 37 degrees for 1 hr, the reaction mixtures induced 34-834 (average 368 +/- 228) revertants per microliter of soy sauce equivalent in Salmonella typhimurium strain TA100 in the absence of S9 mix. The mutagen(s) formed was very unstable under natural daylight and a fluorescent lamp but quite stable under a yellow lamp as well as in the dark. In addition to the known precursors, i.e., tyramine and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, 1-methyl-1,2,3,4-tetrahydro-beta-carboline, which caused weak mutagenesis, was found in the soy sauce. However, the sum of the activities of the three mutagen-precursors after nitrite treatment accounted for only a part of the mutagenicity of nitrite-treated soy sauce. There was in the soy sauce a factor which increased ninefold the mutagenicity of nitrite-treated tyramine, 3-diazotyramine. Therefore, tyramine was considered the principal precursor of the mutagen produced in the nitrite-treated soy sauce. These three precursors together with the mutagenicity augmentation accounted for all the mutagenicity of nitrite-treated sauce. The mutagenicity-augmenting factor in the soy sauce was nonmutagenic before and after nitrite treatment and was stable to heat and light irradiation.

Two fibrous Pseudomonas phages, Pf1 and Pf2, have been isolated and characterized. The phages were serologically related and were indistinguishable morphologically when viewed by electron microscopy. Both phages formed minute turbid plaques. A simple method was devised for easy differentiation of such plaques from the bacterial lawn. The infected bacteria continued to grow and to liberate the phages into the medium. The phages were collected from the turbid “lysate” by salting out and were purified by differential centrifugation and density gradient centrifugation. The purified phages were sensitive to ultrasonics and to a proteolytic enzyme, Nagarse. A rapid lysis mutant was obtained from the progeny of plaquepurified Pf2. Pf2 was spontaneously produced by strain P28 of P. aeruginosa. Neither acridine orange nor antiserum affected the phage-producing capacity of the bacteria.

Mutants that require exogenous 3′,5′-cyclic adenosine monophosphate (cAMP) for exponential growth were isolated from strains deficient in adenyl cyclase. Studies of one strain showed that cAMP is not incorporated into macromolecules; instead, it seems to have a regulatory function, i.e., in media lacking cAMP, cells form ribonucleic acid (RNA) and protein at linear rather than exponential rates. The exact lesion is not known; ribosomes, messenger RNA, and the β and β′ subunits of RNA polymerase continue to be made in absence of added cAMP.

STABLE RNA, including rRNA and tRNA, turns over slowly, if at all, during steady state growth of Escherichia coli1–4. It is evident, however, that E. coli cells can degrade stable RNA, which is slowly broken down to various extents under such conditions as starvation phosphate5,6, magnesium7,8, potassium9 and nitrogen10,11 The mutant described here very rapidly degrades what is essentially all its stable RNA when RNA synthesis is stopped in cultures at 42° C. The degradation can be prevented if antibiotics are added that inhibit breakdown of polysomes.

Summary Pyocin 28 was obtained by induction of Pseudomonas aeruginosa p28 with mitomycin. Pyocin activity was correlated with the number of rod-shaped particles in purified preparations. The width of the pyocin rod was uniform, measuring about 90 A, but the length was not uniform, varying from 200 to 4000 A, but rods measuring 1000 to 1200 A were most frequent. A dark central line and regular cross-striations were usually seen on the rod, and a fine fibre was sometimes visible at the sharp end. Pyocin activity was slightly reduced from ultrasonic treatment, but not at all by trypsin, Nagarse, DNase and RNase. The pyocin was stable between pH 5.0 and 8.0, and was completely inactivated by heating at 60° for 10 min. Specific attachment of numerous pyocin rods to the surface of sensitive bacteria was observed by the electron microscope.

An Escherichia coli mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering str gene (76 min on E. coli linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [3H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.

Heavy-duty diesel-exhaust particles were collected, extracted and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 × g post-mitochondrial supernatant from Aroclor-induced rat liver (S9 mix). The neutral and acidic fractions showed high mutagenicity with TA98 in the absence of S9 mix, the acidic fraction having the highest specific activity. In the absence of S9 mix, the mutagenicity of crude, neutral and acidic fractions was greater in TA98 than in TA98NR and TA98/1,8-DNP6. Chemically-synthesized nitroacetoxypyrenes and nitrohydroxypyrenes were fractionated into the neutral and acidic fractions, respectively. These nitroarenes were purified by high-performance liquid chromatography and their mutagenicity was measured with the 4 strains. With TA98 in the absence of S9 mix, 1-nitro-3-acetoxypyrene, 1-nitro-6/8-acetoxypyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6/8-hydroxypyrene induced 16 700, 336, 992, 94 His+ revertants per plate per nmole, respectively. In the absence of S9 mix, the level of mutagenicity of these nitroarenes was highest in TA98, lowest in TA98/1,8-DNP6 and intermediate in TA98NR. The neutral and acidic fractions of diesel-exhaust particles were analyzed by gas chromatography-mass spectrometry and gas chromatography-mass fragmentography. The neutral fraction was found to contain nitroacetoxypyrenes, 1-nitropyrene, 1,6-dinitropyrene, while nitrohydroxypyrenes were detected in the acidic fraction. The amounts of 1-nitro-3-acetoxypyrene, 1-nitropyrene, 1,6-dinitropyrene and 1-nitro-3-hydroxypyrene were 6.3, 62, 0.81, and 70 ng per mg of crude extract, and accounted for 12, 3.6, 8.0, and 9.0%, respectively, of mutagenicity of the crude extract in TA98 in the absence of S9 mix.

The mutagenic activity of extracts of airborne particulates was evaluated in the Salmonella system. The mutagenicity of airborne particulates was not always correlated with the content of benzo[a]pyrene (B[a]P) in the complex mixtures, especially when the samples were collected at different sites. Large-scale fractionation of extracts of airborne particulates was used to determine the content of specific mutagenic chemicals. The neutral fraction of material soluble in cyclohexane and nitromethane contained the polycyclic aromatic hydrocarbon (PAH) compounds, which accounted for 27.9% of the mutagenic activity of the whole extracts. 9 kinds of PAH compound were identified quantitatively by thin-layer chromatography. They included, per 1000 m3 of air, 12.6 microgram of benzo[e]pyrene (B]e]P), 10.7 microgram of chrysene (CHRY), 10.0 MICROGRAM OF FLUORANTHENE (FL), 6.43 microgram of benzo[ghi]perylene (B[ghi]P), 5.75 microgram of benz[a]anthracene (B[a]A), 5.33 microgram of B[a]P, 3.38 microgram of pyrene (PYR), 1.83 microgram of coronene (COR), and 1.34 microgram of perylene (PERY). Mutagenicity of the ether-soluble acidic, basic and methanol-soluble neutral fractions accounted for 10.9, 9.71 and 6.78% of the total mutagenic activity of crude extract, respectively, when assayed in strain TA98 with liver S9 fraction. The total recovery of mutagenic activity after fractionation was 58%. Two acidic fractions (weak and strong ether-soluble acids) and the methanol-soluble neutral fraction reverted strain TA98 dramatically to prototrophy in the presence of rat-lung S9 fraction more than liver. But the mutagenic chemicals in these fractions remain to be clarified. Direct mutagens were present in essentially all fractions. The particulates, which had diameters ranging from 0.3 to 1.0 micron and were able to penetrate alveoli, contained a high content of mutagens.

Nitrated polycyclic aromatic compounds, 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-diNP), are environmental mutagens and carcinogens. Nitroreductases purified from an anaerobic bacterium, Bacteroides fragilis, catalyzed the metabolic activation of these compounds to produce DNA- and tRNA-bound adducts in vitro. Formation of the adducts was inhibited by p-chloromercuribenzoic acid, which is an inhibitor of nitroreductases from B. fragilis. The enzyme and coenzyme (NADPH) were essential for the adduct formation. These results suggest that nitroreduction is a necessary step in the metabolic activation of nitropyrenes. 1-NP bound specifically to poly(G) and poly(dG), and 1,6-diNP bound to poly(G), poly(dG), and poly(X). The other purine polynucleotides were weak acceptors. However, the reactive products of nitropyrenes formed by nitroreductases could not bind to pyrimidine polynucleotides. Enzymatic hydrolysis of 1-NP-bound DNA and subsequent analysis by high-performance liquid chromatography showed one major and two minor adducts in the hydrolysate. The peak of the major adduct corresponded to that of N-(deoxyguanosin-8-y1)-1-aminopyrene, which is the same as an adduct formed by xanthine oxidase, a mammalian nitroreductase. Nitroreductase activity in the various organs and intestinal contents of Sprague-Dawley rats was assayed in the presence of NADPH or NADH under nitrogen gas. Nitroreductase activity was widely distributed in the organs of the rats; in particular, that of the liver and of the small intestine was relatively high, but that of the respiratory organs such as lung and alveolar macrophages was very low. Intestinal contents had high nitroreductase activity, which was proportional to the number of bacteria, especially anaerobic bacteria, in the intestine. These results suggest that the nitroreductase activity of the normal bacterial flora is very high in rats and that the intestinal bacteria play a major role in the metabolism of nitropyrenes in vivo.

The dose dependencies of the lung carcinogenicity of 1,6-dinitropyrene (1,6-DNP) and benzo[a]pyrene (BaP) were examined by direct injections of these compounds into rat lungs. A total of 276 male F344 rats were divided into 10 groups and given various doses of 1,6-DNP or BaP, or no drug (control group). Both chemicals were injected into the lung, as suspensions in beeswax--tricaprylin and the animals were then observed for 104 weeks. The incidences of lung cancer were 0/39 (0%), 4/30 (13%), 13/31 (42%), 22/26 (85%) and 6/9 (67%) in groups treated with 0.003, 0.01, 0.03, 0.1 and 0.15 mg of 1,6-DNP respectively, and 1/29 (3%), 7/30 (23%), 22/29 (76%) and 9/13 (69%) in those treated with 0.03, 0.1, 0.3 and 1.0 mg of BaP respectively. No lung cancer was found in control rats. Thus the incidences of lung cancer induced by 1,6-DNP and BaP showed significant dose dependence. At equal doses, the incidence of lung cancer was much higher with 1,6-DNP than with BaP, and the induction of cancer by 1,6-DNP was higher even at one-third the dose of BaP. Histologically, most tumors induced by 1,6-DNP were undifferentiated neoplasms, whereas most of those induced by BaP were well-differentiated squamous cell carcinomas.

Nitropyrenes (NPs) are the most potent mutagens that have been detected in environmental pollutants such as airborne particulates (Tokiwa et al., 1981b), car exhaust emissions (Lee et al., 1980; Gibson et al., 1981; Pederson and Siak, 1981; Schuetzle et al., 1981), photocopies (Lofroth et al., 1980; Rosenkranz et al., 1980), wastewater from gasoline stations (Ohnishi et al., 1983; Manabe et al., 1984), and used crankcase oil (Manabe et al., 1984). They are direct-acting mutagens in the Ames Salmonella mutation test; 1-NP, 1,3-diNP, 1,6-diNP, and 1,8-diNP produce 417; 65,500; 82,000; and 100,000 His+ revertants/plate/nmol, respectively, from strain TA98 in the absence of rat liver S9 mix (unpublished data). To induce mutagenicity the NPs are converted to the activated forms by nitroreductases in theSalmonella tester strain (TA98). Since strains TA98NR and TA98/1,8-DNP6 are defective in the specific activating enzymes, they show low mutagenicity by 1-NP and diNPs (Rosenkranz et al., 1981).

Most of the mutagenicity in airborne particulate pollutants is found in respirable particles less than 1 μm in diameter, and polycyclic aromatic hardrocarbons (PAHs) or their derivatives seem to be important mutagens in these particles. In fact, the neutral fraction extracted from airborne particulates contains direct- and indirect-acting mutagens, and accounts for 48% of the mutagenic activity of the whole extract (Tokiwa et al., 1980).

The gene promoting nucleic-acid degradation (pnd) on IncIa plasmid R483 was cloned into pBR322. It is located on a 0.85 kilobase (kb) EcoRI-SalI fragment and is close to Tn7. The pnd gene has similar properties to the srnB gene on the F plasmid. A cleavage map of the 0.85 kb pnd fragment was constructed and compared with that of the 1.18 kb EcoRI-BamHI fragment containing the srnB gene. These two regions showed marked heterogeneity as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution of the two genes for stable RNA degradation.

Air pollution, largely from automobile traffic, has been worsening in the developed countries, especially in urban areas. Since environmental mutagens and carcinogens have come under scrutiny (Menck et al., 1974; Bridges, 1976), a rapid microbial mutation test (Ames et al., 1975) has been applied to the study of air pollution (Chrisp and Fisher, 1980). We reported that the airborne particulates collected in residential and industrial urban areas showed 7-445 h i s + revertants/plate/m 3 of air from tester strain TA98 incubated along with rat-liver extract in the Ames test (Tokiwa et al., 1977). Although we did not know the exact sources of mutagens in the residential area, we pointed out that automobile-exhaust emissions might be the greatest contributor (Ohnishi et al., 1980). In Japan some kinds of pollutants from cars, such as NOx, CO, and hydrocarbons, are subject to control. We also reported that exhaust emissions from an unregulated 1972 car provoked about 12000 h i s + revertants/plate/m 3 of exhaust gas, whereas those from a 'regulated' 1975 passenger car showed about 4000 h i s + rever tants /plate/m 3 from strain TA98 in the presence of S9 mix (Ohnishi et al., 1980). Furthermore, compared to these gasoline engines, automobile exhaust from old diesel engines showed very high mutagenic potency: more than 20000 h i s + rever tants /plate/m 3 (Ohnishi et al., 1980). In West-Germany it has been estimated that 1.85 ton of benzo[a]pyrene was produced by gasoline engines of passenger cars in 1973 (Grimmer and Hildebrandt, 1975). Even when the most restrictive minima were in force on gasoline engines of passenger cars in Japan in 1978, exhaust emissions from a 1978 passenger car (equipped with a catalyst) showed about 1000 h i s + revertants /plate/m 3 in the Salmonella/microsome test (unpublished data); and the emissions of small engines used to generate electric power or to run agricultural machinery are not regulated at all. In this paper we report that particulate exhaust

We have screened 897 temperature sensitive growth mutants of E. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42 degrees C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42 degrees C). Eight stains showed altered turnover of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42 degrees C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42 degrees C, and protein synthesis continued at a significant, but slightly reduced, rate at 42 degrees C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.

Degradation of otherwise stable rRNA and tRNA takes place in the presence of rifampin, dependent on the F plasmid srnB gene. We have reported that a protein newly synthesized in the presence of rifampin might be a product of the srnB gene required for stable RNA degradation (Ito, R. and Ohnishi, Y. (1983) Biochim. Biophys. Acta 739, 27–34). Here we have further studied the mechanism of srnB expression. Among eighteen mutants with altered RNA polymerase, two (TJ2470 ( rpoC4 ) and TJ302 ( rpoC56 )) showed RNA degradation at high temperature (42°C) when the srnB gene was present. Labeling proteins at 42°C in strain TJ2470 indicated that a protein of molecular weight 12 000 was a product of the srnB gene, and that expression of the srnB gene provoked RNA degradation. Using plasmid pTK4, in which the srnB gene is inserted downstream of the promoter of lacZ, lac promoter-dependent expression of the srnB gene, with production of the putative protein product, also induced RNA degradation at 42°C, with no requirement for added rifampin or altered RNA polymerase. RNA degradation in these conditions was quite similar to that in the case of the addition of rifampin; e.g., it showed some responses to Mg 2+ , temperature and RNAase I content of the cells. Expression of the srnB gene dependent on lac promoter was also observed in minicells. Thus, it is inferred that the srnB gene is probably repressed under normal conditions with its own promoter; its expression initiates RNA turnover.

Ames's strains of Salmonella typhimurium were used to evaluate the mutagenic activity of airbone particulate materials collected at six different points in the industrial area of Ohmuta and the residential area Fukuoka. Tests were done in presence of rat-liver S-9 fraction isolated from rats that had been treated with Aroclor 1254. When the number of revertant colonies per plate was plotted against the amount of methanol extract of particulate air pollutants, using strain TA98, approximately linear relationships were observed for active samples. Generally, mutagenic activity of the samples increased in proportion to the density of air pollutants. In our system, 38--349 microng of methanol extract, from 0.225--4.51 m3 of air from the factory districts in Ohmuta City gave 100 his+ revertants per plate. On the other hand, 54--2300 microng of air pollutants, from 1.29--14.1 m3 of air from the residential districts in Fukuoka City, gave a comparable activity. Every sample from each area had mutagenic activity. Chemicals in air pollutants were fractionated by alumina column chromatography and identified by gas chromatography and mass spectrometry. More than 28 compounds, including 12 unknown substances were identified as polycyclic hydrocarbons. Twelve of these compounds are already known to be carcinogens and to induce reversions to histidine independence in strain TA98 of Salmonella.

In tests on the carcinogenicity of 1,6-dinitropyrene [(1,6-DNP) CAS: 42397-64-8] and 1-nitropyrene [(1-NP) CAS: 5522-43-0], 0.1 mg of each compound was inoculated sc into BALB/c mice once a week for 20 weeks. In the group given injections of 1,6-DNP the first tumor appeared on day 112, and 10 of the 20 mice developed tumors at the injection site by 45 weeks after the first injection. However, no tumors were induced in any of the mice that received injections of 1-NP. All of the induced tumors were transplantable for more than five generations in male BALB/c mice. Most of the tumors showed the characteristic histologic features of malignant fibrous histiocytoma.

The pnd gene of R plasmid R483, like the srnB gene of the F plasmid, increases the degradation of stable RNA in Escherichia coli. The nucleotide sequence of the pnd locus was determined and compared with that of the srnB locus. The genes have open reading frames that are 54% homologous, and both have an upstream inverted repeat sequence. The pnd gene expression seems to decrease the osmotic barrier of the cytoplasmic membrane, since no plasmolytic vacuoles were formed in the cells carrying the gene when the cells were exposed to hypertonic sucrose solution. This result suggests that RNase I in the periplasm passes through the altered membrane to degrade stable RNA in the cytoplasm.

Modem industry has produced many useful chemical agents, but they and their by-products have also contributed to environmental pollution. The effects of environmental chemicals on human health are of considerable concern, since damage to DNA by environmental mutagens, both natural and man-made, is likely to be a major cause of cancer and other diseases (Rosenkranz, 1973; Doll, 1977; Wynder and Gori, 1977; Ames, 1979).

Genotoxicity of a variety of nitroarenes and other compounds was examined in DNA-repair tests with rat or mouse hepatocytes. Out of 15 nitroarenes tested, 9 compounds, i.e., 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, 1-nitro-3-acetoxypyrene, 3-nitrofluoranthene, 2-nitrofluorene, 2,7-di-nitrofluorene and 5-nitroacenaphthene elicited positive response of DNA repair in the tests with rat and mouse hepatocytes. Among the positive chemicals, the DNA-repair level of the 3 dinitropyrene isomers was much higher than other nitroarenes. The results indicate that a number of nitroarenes are metabolically activated in the primary culture of rodent hepatocytes, and suggest potential carcinogenicity of 1-nitropyrene and 1-nitro-3-acetoxypyrene the carcinogenicity of which is either not clear or unknown. Of the other nitro compounds, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide as well as 4-nitroquinoline 1-oxide were clearly genotoxic in the assays with hepatocytes of both species. However, 5-nitro-2-furaldehyde semicarbazone was negative in both assays with hepatocytes of 2 species.

The gene promoting nucleic-acid degradation (pnd) of IncB plasmid R16 was cloned into the vector plasmid pACYC177. The pnd gene was found to be located on a 0.55-kilobase (kb) AluI-PstI fragment by constructing subclones carrying various portions of the initially cloned fragment. The direction of transcription of the pnd gene was determined by inserting the gene in both orientations into the lacZ′ gene of the plasmid pUR222. In the recombinant plasmid pCM2, transcription of the pnd gene was controlled by the lac promoter region. Addition of cAMP at 42 C resulted in rapid degradation of stable RNA in cells harboring pCM2. In contrast, no RNA degradation was observed in cells harboring pCM14, which has the same insert as pCM2 but in the opposite orientation. The equivalent gene, pnd of IncIα plasmid R483, has previously been cloned, and a detailed restriction map of the region has been constructed (Akimoto, S., and Ohnishi, Y. 1982. Microbiol. Immunol. 26: 779–793). We constructed a detailed restriction map of the pnd region of R16 and compared it with that of R483. Restriction analyses revealed a similar structure in these two pnd regions. The results suggest that the pnd genes of R16 and R483 have a common evolutional origin.

Mutagenic nitro derivatives were readily induced when 6 kinds of chemicals were exposed to 10 ppm of nitrogen dioxide (NO2). Single nitro derivatives were formed from pyrene, phenanthrene, fluorene or chrysene. Carbazole and fluoranthene each produced 2 derivatives substituted with nitro groups at different positions. The formation of nitro derivatives was enhanced by exposure of pyrene to NO2 containing nitric acid (HNO3, less than 100-fold enhancement) or sulphur dioxide (SO2, less than 15-fold enhancement). After 24 h of exposure the yields of the nitro derivative were 0.02% with 1 ppm of NO2 in air and 2.85% with NO2 (1 ppm) containing traces of HNO3. The nitro derivatives from all but phenanthrene and carbazole were chemically identified by means of gas chromatography (GC) and mass spectrometry (MS), and the mutagenicity of the 4 kinds of authentic nitro derivatives was tested by using Salmonella strains TA98 and TA1538 with or without the S9 fraction from rat liver treated with Aroclor 1254. The nitro derivative induced from pyrene was determined to be 1-nitropyrene; that of chrysene was 6-nitrochrysene; that of fluorene was 2-nitrofluorene; and those of fluoranthene were 3-nitrofluoranthene, and 8-nitrofluoranthene. Tested with strain TA98 in the absence of the S9 fraction, the first 4 of these derivatives yielded, respectively, 3050, 269, 433 and 13 400 revertants per nmole. Thus, each nitro derivative formed was potentially a direct-acting frameshift-type mutagen. Each compound exposed to NO2 showed a decreased mutagenic activity when tested in the presence of S9 mix. A possible explanation comes from experiments in which 1-nitropyrene was incubated with the S9 mix at 37 degree C for 10 min, and 1-aminopyrene was formed. The mutagenic activity of 1-aminopyrene was appreciable, but only about one-tenth of that of 1-nitropyrene in the Ames test.

The disparity between the known roles of cyclic AMP (cAMP) in bacteria and higher cells has been puzzling. In mammalian cells, as participants of this Symposium have been reminding us, a number of effects are exerted independent of the transcription process, presumably by activating latent enzymatic machinery-or possibly latent messenger RNA. In contrast, for the best characterized systems in bacteria, cyclic AMP helps to determine the repertoire of gene transcription open to the cell [1–5].

Unlike mutations to streptomycin resistance, which restrict the function of an SU II amber suppressor, mutations to spectinomycin resistance have no restrictive effect. Furthermore, mutations to spectinomycin resistance counteract the restrictive action of streptomycin-resistant mutations when combined in a single strain. The mutations to drug resistance, singly or in combination, do not themselves lead to any detectable suppressor activity. The effects on SU II are interpreted in terms of interactions between ribosomal elements.

The mutagenicity of cooked foods was investigated by using the market basket samples collected for a total diet study. Eighty-nine foods were divided into thirteen groups and cooked. These cooked food groups were treated with amyloglucosidase, hesperidinase, pectinase or papain, and extracted with methanol. The crude methanol extracts were successively extracted with n-hexane and diethyl ether under acidic conditions and also extracted with diethyl ether under basic conditions. The mutagenicity of each extract was assayed by means of the Ames test.All food groups except the rice group, and the fat and oil group were mutagenic in Salmonella typhimurium strain TA98 or TA100 with or without S9mix. Quercetin was detected in some groups by gas chromatography-mass spectrometry, but other kinds of mutagens in the cooked foods were not detected. According to this total diet study, Japanese people daily ingest mutagenic substances, which are mainly derived from fruits (33% of the total mutagenic activity for strain TA100 in the presence of S9mix), fish and shellfish (24%), pulses (15%), and meats (13%).