Yoshinari Ohnishi

Mutagenicity and Carcinogenicity of Nitroarenes and Their Sources in the Environment

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.

Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide

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.

Mutagenic and chemical assay of extracts of airborne particulates

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.

Triterpenes augment the inhibitory effects of anticancer drugs on growth of human esophageal carcinoma cells in vitro and suppress experimental metastasis in vivo

The antineoplastic effects of combinations of anticancer drugs (5‐fluorouracil, irinotecan and cisplatin) and triterpenes (ursolic acid, betulinic acid, oleanolic acid and a Japanese apricot extract (JAE) containing triterpenes) on esophageal squamous carcinoma cells were examined by the WST‐8 (2‐(2‐methoxy‐ 4‐nitrophenyl)‐3‐(4‐nitrophenyl)‐5‐(2,4‐disulfophenyl)‐2H‐tetrazolium, monosodium salt) assay in vitro and by an animal model in vivo. Triterpenes and JAE showed additive and synergistic cytotoxic effects, respectively, on esophageal squamous carcinoma cells (YES‐2cells) by combinational use of 5‐fluorouracil. JAE and 5‐fluorouracil induced cell cycle arrest at G2/M phase and at S phase, respectively, and caused apoptosis in YES‐2 cells. A new animal model of esophageal cancer causing tumor colonization of the peritoneal cavity and producing bloody ascites was made by injecting YES‐2 cells into the peritoneal cavity of a severe combined immunodeficiency mouse. In this model, 5‐fluorouracil inhibited colonization of tumor cells in the peritoneum. The addition of JAE to 5‐fluorouracil augmented the suppression of experimental metastasis of the peritoneum. The numbers of peritoneal nodules of more than 2 mm in diameter in mice treated with 5‐fluorouracil and JAE were less than those in mice treated with 5‐fluorouracil alone or JAE alone. These results suggest that triterpenes, especially JAE, are effective supplements for enhancing the chemotherapeutic effect of 5‐fluorouracil on esophageal cancer.

Detection of 1-nitropyrene in yakitori (grilled chicken)

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)

Gene affecting longevity of messenger RNA: A mutant ofEscherichia coli with altered mRNA stability

Summarywe have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over 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° 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° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° 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.

Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice.

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.

Role of Intestinal Bacteria in Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug

The role of intestinal bacteria in induction and repression of ulcer formation in the ileum of rats treated with one of the nonsteroidal antiinflammatory drugs (NSAIDs), 5‐bromo‐2‐(4‐fluorophenyl)‐3‐(4‐methylsulfonylphenyl) thiophene (BFMeT), was examined in this study. BFMeT was administered by intragastric gavage once at doses of 500‐1,500 mg/kg of body weight to Wistar rats treated with and without antibiotics (bacitracin, neomycin, streptomycin), germ‐free rats and gnotobiotic rats, and 72 hr later their gastrointestinal tracts were examined for ulcer formation. A single oral administration of BFMeT induced ileal ulcers in specific pathogen‐free rats. However, the rats given antibiotics to reduce the intestinal bacteria had no ulcers. BFMeT‐treated germ‐free rats and gnotobiotic rats mono‐associated with Bifidobacterium adolescentis or Lactobacillus acidophilus also had no intestinal ulcers. However, the drug induced ileal ulcers in gnotobiotic rats mono‐associated with Eubacterium limosum or Escherichia coli. An overnight culture of B. adolescentis or L. acidophilus or yogurt containing Bifidobacterium breve and Streptococcus thermophilus, when given as drinking water, inhibited ulcer formation in the ileum of rats treated with BFMeT. Gram staining of the ileal contents of normal rats revealed that 97.4% of the stained microorganisms were Gram‐positive rods and only 1.2% were Gram‐negative rods. In the group of rats with ulcers induced by BFMeT, the Gram‐positive rods decreased by 56.4% and the Gram‐negative rods including Escherichia coli, Klebsiella, Proteus and Bacteroides increased by 37.3%. However, in the group of rats administered the Bifidobacterium culture, the Lactobacillus culture or yogurt, the percentages of the Gram‐negative rods were decreased. Although Lactobacillus was a major bacterium in the ileum of normal rats, the Gram‐negative facultatively anaerobic rods E. coli, Klebsiella and Proteus were increased in the ulcerated ileum of rats treated with BFMeT, suggesting that these bacteria are associated with ulcer formation in rats treated with NSAIDs, and that Lactobacillus and Bifidobacterium inhibit it by repressing the growth of ulcer‐inducing bacteria.

Identification and quantification of highly mutagenic nitroacetoxypyrenes and nitrohydroxypyrenes in diesel-exhaust particles.

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 X 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.

Detection of mutagenic activity in automobile exhaust.

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/m3 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/m3 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/m3 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/m3 toward strain TA98 and 87 toward strain TA100. The ether-soluble neutral fraction yielded 86 revertants/plate/m3 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/m3 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.