1-nitropyrene-4-5-oxide and 1-nitropyrene-9-10-oxide

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.

Topics: Base Sequence; Cell Line, Transformed; DNA; Fibroblasts; Genes, Suppressor; Genetic Vectors; Humans; Molecular Sequence Data; Mutagens; Mutation; Plasmids; Pyrenes; RNA, Transfer; Xeroderma Pigmentosum; Xeroderma Pigmentosum Group A Protein

Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were A-->G transition (15/33) and G-->A transition (8/33). The predominant base substitution, A-->G transition (11/27) and G-->A transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.

Topics: Animals; Base Sequence; CHO Cells; Cricetinae; Gene Deletion; Hypoxanthine Phosphoribosyltransferase; Mutagenicity Tests; Mutagens; Point Mutation; Pyrenes; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA

To elucidate the effects of the intestinal microflora on absorption and activation of glutathione conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), we investigated the biological activities of the microflora in specific-pathogen-free (SPF) mice and SPF mice treated with various antibiotics and established the methodology of antibiotic treatment to eliminate the intestinal microflora. Mice were given various kinds of antibiotics by intragastric gavage twice a day for five days. A mixture of antibiotics bacitracin (BC), neomycin (NM) and streptomycin (SM) was the most effective in reducing the various activities of the intestinal microflora. The treatment decreased the bacterial counts and the activities of enzymes of the intestinal contents cysteine conjugate beta-lyase (beta-lyase), beta-glucuronidase and nitroreductase which were derived from the intestinal microflora, but did not affect the activities of gamma-glutamyltransferase and aminopeptidase which were derived from host tissue cells. Furthermore, the treatment did not affect absorption of glucose from the intestinal tract, body weight or liver enzyme activities. The treatment with only an aminoglycoside antibiotic, kanamycin or NM, decreased neither the number of anaerobes in the intestine nor the beta-lyase or nitroreductase activities from the intestinal contents. Glutathione conjugates of [3H]-1-NP oxides were administered to two groups of ICR mice that had been treated with antibiotics (BC, NM, SM) or saline (control group) orally. The radioactivity in the blood increased and reached the maximum level 2 or 3 h after administration of the conjugates in the control group; however, that in the antibiotic-treated group was only slightly increased if at all. Excretion of [3H]-labeled metabolites into the urine was approximately 20% of the total dose in the control group, but it was < 2% in the antibiotic-treated group during 48 h. After 48 h, DNA in the lower intestinal mucosa was extracted and the DNA adducts were analyzed by the 32P-postlabeling method. Three new DNA adducts were detected in the lower intestinal mucosa of the control group but not of the antibiotic-treated group. These results suggest that the intestinal microflora plays an important role in absorption of the metabolites of glutathione conjugates of 1-NP oxides from the intestinal tract and activation of the metabolites in the intestine.

Topics: Animals; Anti-Bacterial Agents; Aryl Hydrocarbon Hydroxylases; Bacitracin; Biotransformation; Carbon-Sulfur Lyases; DNA; Drug Therapy, Combination; Glucuronidase; Glutathione; Intestinal Absorption; Intestinal Mucosa; Kanamycin; Liver; Lyases; Male; Mice; Mice, Inbred ICR; Neomycin; Nitroreductases; Phosphorus Radioisotopes; Pyrenes; Streptomycin; Time Factors; Tritium

DNA adduct formation in the liver of B6C3F1 mice after administration of 1-nitropyrene (1-NP) was shown by the 32P-postlabeling technique. The major adduct was not N-(deoxyguanosin-8-yl)-1-aminopyrene, which was easily formed in in vitro nitroreduction of 1-NP in the presence of DNA, but the major spots migrated to the same position as the in vitro DNA adduct spots of K-region epoxides of 1-NP (1-NP 4,5- and 9,10-oxide). 1-NP oxides formed by the oxidative activation of 1-NP in the liver were excreted into the bile as detoxified glutathione conjugates which were changed to cysteine conjugates in the upper intestinal tract. The cysteine conjugates were degraded by cysteine conjugate beta-lyase (beta-lyase) of intestinal microflora in the lower intestinal tract. The mutagenicity of cysteine conjugates of 1-NP oxides for Salmonella typhimurium was enhanced by addition of beta-lyase and was decreased by addition of aminooxyacetic acid, a beta-lyase inhibitor. The in vitro binding of the cysteine conjugates to calf thymus DNA was increased by addition of beta-lyase and xanthine oxidase. We administered glutathione conjugates of 1-NP oxides to two groups of mice that had been treated with antibiotics or saline by gavage and analyzed the DNA adducts in the lower intestinal mucosa. The specific DNA adducts were detected in the saline-treated group but not in the antibiotics-treated group. These results suggest that intestinal microflora play an important role in activation of glutathione conjugates of 1-NP oxides.

Topics: Animals; Bacteria; DNA; Glutathione; Intestines; Liver; Lyases; Mice; Mice, Inbred Strains; Pyrenes

The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol/HQ, even when measured by the standard and modified nuclease P1 postlabeling methods capable of detecting 1 adduct in 10(9-10) DNA bases.

Topics: Acid Phosphatase; Adenosine Triphosphate; Animals; Benzoquinones; DNA; Female; Hydroquinones; Nucleosides; Nucleotides; Phenol; Phenols; Phosphorus Radioisotopes; Pyrenes; Quinones; Rats; Rats, Inbred Strains; Single-Strand Specific DNA and RNA Endonucleases

[4,5,9,10-(3)H]1-Nitropyrene was incubated with liver microsomes prepared from guinea pigs treated with Aroclor 1254 and the products were examined by h.p.l.c. The previously reported metabolites, 1-nitropyrene trans-4,5-dihydrodiol, 1-nitropyrene trans-9,10-dihydrodiol, and 3-, 6-, and 8-hydroxy-1-nitropyrene were detected. In addition, h.p.l.c., nuclear magnetic resonance and mass spectral analyses indicated the presence of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide. The epoxide hydrase inhibitor, 1,2-epoxy-3,3,3-trichloropropane, decreased the concentration of the 4,5- and 9,10-dihydrodiols in the microsomal incubations and increased the concentration of their corresponding oxides. Reaction of 1-nitropyrene with m-chloroperoxybenzoic acid gave a mixture of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, which was separated by chromatography. The mutagenicity of the oxides was determined in Salmonella typhimurium strains TA98, TA98NR, and TA98/1,8-DNP6, both with and without exogenous activation by a rat liver homogenate fraction (S9). In the absence of S9, both oxides showed maximum activity in TA98, slightly decreased mutagenicity in the acetylase-deficient strain TA98/1,8-DNP6, and much reduced activity in the nitroreductase-deficient strain, TA98NR. When assayed in the presence of S9, 1-nitropyrene 4,5-oxide had maximum mutagenicity in TA98, and was 50 and 95% less mutagenic in TA98NR and TA98/1,8-DNP6, respectively. 1-Nitropyrene 9,10-oxide had a similar strain sensitivity, except that its total mutagenicity was lower. Since 1-nitropyrene is metabolized by oxidative pathways in vivo, these K-region oxides may contribute to the toxicities elicited by this compound.

Topics: Animals; Biotransformation; Epoxide Hydrolases; Guinea Pigs; In Vitro Techniques; Male; Microsomes, Liver; Mutagenicity Tests; Mutagens; Pyrenes; Rats; Rats, Inbred Strains; Salmonella typhimurium; Spectrum Analysis; Trichloroepoxypropane