phosphorus-radioisotopes and 1-nitropyrene-4-5-oxide

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

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