Takemi Kinouchi

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)

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.

Intestinal anaerobic bacteria hydrolyse sorivudine, producing the high blood concentration of 5-(E)-(2-bromovinyl)uracil that increases the level and toxicity of 5-fluorouracil.

Sorivudine, 1-beta-D-arabinofuranosyl-5-(E)-(2-bromovinyl)uracil, is a potent antiviral agent against varicella-zoster virus and herpes simplex virus type 1. However, sorivudine should not be used in combination with anticancer drugs such as 5-fluorouracil (5-FU) because (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of sorivudine, inhibits the degradation of 5-FU, resulting in its accumulation in the blood and marked enhancement of the toxicity of 5-FU. Since phosphorolytic enzymes generate BVU from sorivudine, we investigated the distribution of the enzyme activity in rats. High activity was found in the cecal and large intestinal contents, while very low or no detectable activity in the liver, kidney, stomach, cecum, large intestine, and the stomach and small intestinal contents. These results suggest that intestinal microflora play an important role in BVU production. Therefore, we measured the phosphorylase activity in cell-free extracts from 23 aerobes, 16 anaerobes and a fungus. Bacteroides species B. vulgatus, B. thetaiotaomicron, B. fragilis, B. uniformis and B. eggerthii, dominant members of intestinal microflora, had high activity to convert sorivudine to BVU. To elucidate the contribution of intestinal microflora to BVU production in vivo, we administered sorivudine to rats treated with several antibiotics and measured the BVU concentration in the serum of rats. When sorivudine was given to rats treated with ampicillin or a mixture of bacitracin, neomycin and streptomycin, which decreased the numbers of viable aerobes and anaerobes, only a small amount of BVU was found in the serum. BVU concentration in the serum of rats treated with metronidazole to decrease the number of intestinal anaerobes was also very low. In contrast, BVU concentration in the serum of rats treated with kanamycin, which was used to decrease the number of aerobes selectively, was higher than that of non-treated rats. These results also suggest that BVU is produced by intestinal anaerobic bacteria especially Bacteroides species in vivo.

Biotransformation of 1-nitropyrene in intestinal anaerobic bacteria.

Mutagenic nitroaromatic compounds have recently been found in photocopies, urban atmosphere, automobile exhaust and wastewater. 1‐Nitropyrene (1‐NP) is readily formed when pyrene, ubiquitous in the environment, is exposed to nitrogen dioxide in the urban atmosphere or in automobile exhaust, and is highly mutagenic, inducing 449 his+ revertants/plate/nmol from Salmonella typhimurium strain TA98 in the absence of S9 fraction in the Salmonella‐microsome test. It is possible to swallow sputum or some food containing 1‐NP and it would come into contact with the normal bacterial flora. We determined the 1‐NP nitroreductase activity in environmental and laboratory bacterial strains. We found that the mutagenicity of 1‐NP mixed with the feces of a healthy man or a culture of anaerobic bacteria was decreased. The product proved to be 1‐aminopyrene (1‐AP), based on its fluorescence spectrum, its mass spectrum, and its characteristic thin layer chromatographic and high performance liquid chromatographic patterns. The 1‐NP nitroreductase activity of aerobic bacteria was low, but crude extracts from the anaerobic bacteria, i.e., Bacteroides fragilis, B. thetaiotaomicron, B. vulgatus, Fusobacterium mortiferum, F. nucleatum, Clostridium perfringens, C. sporogenes, Bifidobacterium adolescentis, B. bifidum, Eubacterium lentum, E. limosum, and Peptostreptococcus anaerobius, all easily converted 1‐NP to 1‐AP and proportionally decreased the mutagenic activity of 1‐NP.

Culture Supernatants of Lactobacillus acidophilus and Bifidobacterium adolescentis Repress Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug by Suppressing Unbalanced Growth of Aerobic Bacteria and Lipid Peroxidation

A nonsteroidal antiinflammatory drug, 5‐bromo‐2‐(4‐fluorophenyl)‐3‐(4‐methylsulfonylphenyl) thiophene (BFMeT), induced ileal ulcers in rats after oral administration, while no ulcers were observed after subcutaneous injection. The ileal ulcer formation in BFMeT‐treated rats was examined to correlate the administration of cultures of Lactobacillus acidophilus or Bifidobacterium adolescentis with intestinal bacteria in the ileal contents and lipid peroxidation of the small intestinal mucosa. Ileal ulcers were observed in more than 85% of the rats treated with BFMeT at a dose of 1,000 mg/kg when they were given tap water as drinking water. The incidence of ulcer formation was repressed by giving culture supernatants of L. acidophilus or B. adolescentis as drinking water, but not by giving the cell suspension as drinking water. Gram staining of the ileal contents of normal rats revealed that 97% of the stained bacteria were Gram‐positive rods and only 1.5% were Gram‐negative rods. The percentage of Gram‐negative rods 72 hr after BFMeT administration was 49.8% and increased over 30‐fold in BFMeT‐treated rats. However, the percentage of Gram‐negative rods was 9.7% or 16%, respectively, in rats taking culture supernatants of L. acidophilus or B. adolescentis. In addition, thiobarbituric acid‐reactive substances in the ileal mucosa increased significantly in the rats given tap water for 72 hr after BFMeT treatment, but not in rats given the culture supernatants of L. acidophilus or B. adolescentis. Since BFMeT induced an unbalanced intestinal microflora, the effect of antibiotic treatment on ulcer formation in rats was examined. The magnitude of the ulcer formation in the antibiotic‐treated rats was, in decreasing order, metronidazole >none > kanamycin > a mixture (bacitracin, neomycin and streptomycin). These results suggest that the intestinal microflora plays an important role in ulcer formation and that a metabolite(s) of L. acidophilus and B. adolescentis inhibits ileal ulcer formation by repressing changes in the intestinal microflora and lipid peroxidation in BFMeT‐treated rats.

Effects of dietary bile acids on formation of azoxymethane-induced aberrant crypt foci in F344 rats.

The present study has demonstrated the influence of bile acids (BAs) on the development and growth of azoxymethane (AOM)-induced aberrant crypt foci (ACF). Male F344 rats were treated with two doses of AOM (15 mg/kg) at 7 days apart and fed either basal MF or MF plus 0.4% of cholic (CA), deoxycholic (DCA), chenodeoxycholic (CDCA), lithocholic (LCA) and ursodeoxycholic (UDCA) acid mixed diets for 8 weeks after the first AOM dose. The mean number of ACF/colon of the rats fed CA, DCA, CDCA and LCA were higher than that of MF-fed group and the differences were statistically significant (P < 0.005). But the mean number of ACFs/colon was significantly (P < 0.005) lower in UDCA diet-fed rats compared to MF. UDCA-fed rats also showed a significant decrease in average crypt multiplicity (number of crypts/focus) of ACF compared to MF alone. The mean number of ACF with > or =5 crypts was about 2.5-3.7 times higher in case of CA, DCA, CDCA and LCA and about 8.2 times lower in UDCA compared to the control MF diet group. In a parallel study, feeding for 18 weeks of the same BAs mixed diets without AOM administration did not significantly induce ACF. Therefore, these data suggest that dietary BAs by themselves do not induce ACF in F344 rats but enhance or, in the case of UDCA, suppress the development and growth of AOM-induced ACF.

Nitro Compounds in Environmental Mixtures and Foods

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).

Protective Effects of a Culture Supernatant of Lactobacillus acidophilus and Antioxidants on Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug

Ileal ulcers and thiobarbituric acid (TBA)‐reactive substances in the ileal mucosa were induced in rats treated with a nonsteroidal antiinflammatory drug, 5‐bromo‐2‐(4‐fluorophenyl)‐3‐(4‐methylsulfonylphenyl)thiophene (BFMeT), at a dose of 1,000 mg/kg administered with tap water as drinking water. However, the formation of ileal ulcers and TBA‐reactive substances in the ileal mucosa was repressed by giving the animals a culture supernatant of Lactobacillus acidophilus as drinking water. We measured the antioxidative activity of the culture supernatant and found that the supernatant inhibited the formation of t‐butyl hydroperoxide‐induced TBA‐reactive substances in erythrocyte membrane ghosts. Therefore, the effects of various known antioxidative compounds on the ileal ulcer formation induced by BFMeT were investigated. While α‐tocopherol, t‐butyl‐1,4‐hydroxyanisole and allopurinol did not repress ulcer formation after BFMeT treatment, ascorbic acid, dimethyl sulfoxide, glutathione and β‐carotene significantly inhibited formation. Among these compounds, ascorbic acid was the most effective. Accumulation of TBA‐reactive substances in the ileal mucosa after BFMeT treatment also decreased significantly in rats treated with ascorbic acid. In addition, the percentage of Gram‐negative rods in the ileal contents of rats treated with BFMeT and tap water was dramatically increased, but it was not increased in rats treated with BFMeT and these antioxidants. A positive correlation between the percentage of Gram‐negative rods and the number of ileal ulcers was also observed. These results suggest that lipid peroxidation mediated by oxygen radicals plays an important role in the induction of ileal ulcers by BFMeT in rats, and that lipopolysaccharide‐activated neutrophils probably produce highly reactive hypochlorous acid and hydrogen peroxide, which are inactivated by ascorbic acid and glutathione, respectively.

Isolation and Partial Characterization of Exosporium from Spores of a Highly Sporogenic Mutant of Clostridium botulinum Type A

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine‐HCl and almost completely disrupted with 8 M urea in combination with 1 % mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1 %) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.