Coordinate expression of inducible nitric oxide synthase and cyclooxygenase-2 genes in uterine tissues of endotoxin-treated pregnant mice.

OBJECTIVES: Our purpose was to investigate the relationship between expression of cyclooxygenase-2 and inducible nitric oxide synthase genes after labor induction with bacterial lipopolysaccharide in a murine model of preterm parturition. STUDY DESIGN: Pregnant C57B1/6 mice were given Escherichia coli lipopolysaccharide (20 micrograms per mouse) by intraperitoneal injection on day 16 of gestation, and the animals were followed up for signs of labor. Control mice received an equivalent volume of 0.9% saline solution. The latency from lipopolysaccharide injections until appearance of the first pup was recorded. Two separate groups of mice were given either aminoguanidine or indomethacin (5 mg/kg intragastric) 24 hours before induction of preterm labor. In a separate set of experiments mice were treated with lipopolysaccharide as described and were killed at intervals from 0.5 to 72 hours and intrauterine tissues (uterus, placenta, and fetal membranes) were removed and snap frozen in liquid nitrogen. Total protein and ribonucleic acid were extracted for Western and Northern blot analysis of cyclooxygenase-2 and inducible nitric oxide synthase protein and messenger ribonucleic acid, respectively. RESULTS: Northern blots from uterine, placental, and fetal membrane tissues of lipopolysaccharide- and saline solution-treated mice revealed that cyclooxygenase-2 and inducible nitric oxide synthase messenger ribonucleic acid transcripts were rapidly (within 0.5 to 2 hours) up-regulated after lipopolysaccharide administration but were unchanged in mice injected with saline solution. Immunoblot analysis with isoform-specific antibodies revealed that both enzymes were expressed in uterus, placenta, and fetal membranes in a coordinated fashion with peak expression seen at 6 to 8 hours. Although the steady-state accumulation of messenger ribonucleic acid transcripts encoding cyclooxygenase-2 and inducible nitric oxide synthase peaked at 6 hours and declined to baseline by 16 hours after injection with lipopolysaccharide, expression of cyclooxygenase-2 and inducible nitric oxide synthase was sustained through the period when premature delivery was observed. Nitric oxide-dependent cyclooxygenase-2 and inducible nitric oxide synthase expression was demonstrated by the elimination of accumulation of both messenger ribonucleic acid transcripts in mice pretreated with aminoguanidine before injection with lipopolysaccharide. CONCLUSIONS: These data indicate that nitric oxide synthesis may be a prerequisite for subsequent stimulation of cyclooxygenase-2 and inducible nitric oxide synthase gene expression. Taken together, the data suggest that cyclooxygenase-2 and inducible nitric oxide synthase are expressed in a coordinated manner in the uterus of endotoxin-challenged pregnant mice and that their enzymatic products may contribute to the signaling of uterine activity or cervical changes culminating in expulsion of the fetus.

Application of the 32P-postlabelling assay to the inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adduct formation by dietary fatty acids.

The potent heterocyclic food mutagen IQ is carcinogenic in the CDF1 mouse, affecting the liver, lung and forestomach. Using 32P-postlabelling methods we have isolated up to five IQ-DNA adducts from both target and non-target organs after oral administration of IQ. Up to four additional non-specific adducts, found when the 32P-postlabelling assay was run under intensification conditions, could be attributed to the use of a certain brand of microcentrifuge tube in the assay. CLA is a mixture of heat-generated derivatives of linoleic acid, each with a conjugated double bond system, that has chemopreventive properties in rodents. To examine the effect of CLA on the formation of IQ-DNA adducts in CDF1 mice, we administered CLA by gavage every other day for 45 days, followed by a single oral dose (50 mg/kg) of IQ. Tissues collected 24 h later were analysed for IQ-DNA adducts by 32P-postlabelling. Compared to controls, CLA treatment caused inhibition of adduct formation in the liver, lung, large intestine and kidney. In the kidneys of females, CLA treatment inhibited IQ-DNA adduct formation almost completely (95.2%). Male F344 rats were fed a control diet or an isocaloric diet containing 20% menhaden oil (MO), a rich source of omega-3 fatty acids, for six weeks, then given a single oral dose of IQ. Analysis for IQ-DNA adducts by 32P-postlabelling one or six days later revealed that on day 1 the MO diet caused a 6-7 fold decrease in adduct formation in the liver and an up to 2-fold decrease in both the small and large intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adduct formation in CDF1 mice by heat-altered derivatives of linoleic acid.

Grilled ground beef contains a number of carcinogens, including aminoimidazoazaarenes, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as well as anticarcinogenic substances, such as heat-generated derivatives of linoleic acid (CLA). In the present study, CLA was administered by gavage every other day to young adult CDF1 mice for a period of 45 days (50 microliters/48 hr for days 1-24 and 100 microliters/48 hr for days 25-45), using trioctanoin as a control. On day 46 all animals received a single oral dose (50 mg/kg) of IQ and tissues were collected 24 hr later. Tissue DNA was purified and analysed for IQ-DNA adducts by 32P-postlabelling assays. Compared with controls, CLA treatment caused a 43.1 and 31.8% inhibition of adduct formation in the livers of male and female mice, respectively. In the lung and large intestine CLA had a 74.2 and 39.4% inhibitory effect, respectively, in the female only, whereas there was no effect in the stomach or small intestine of either sex. In the kidneys of females, CLA treatment inhibited IQ-DNA adduct formation almost completely (95.2%), whereas in the kidneys of males CLA had no effect. It is concluded that CLA inhibits IQ-DNA adduct formation in certain IQ target organs (liver and lung) and non-target organs (large intestine, kidney), but is inactive in other target organs (stomach) and non-target organs (small intestine) of the CDF1 mouse.

Sex differences in the formation and persistence of DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in CDF1 mice.

The potent food mutagen 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) is carcinogenic in the CDF1 mouse, affecting the liver, lungs and forestomach. Females are approximately twice as sensitive as males to liver tumor induction. Using 32P-postlabeling assays, the dose-response of IQ-DNA adduct formation was determined in various organs of male and female CDF1 mice after single p.o. doses of IQ. To determine the possible correlation between IQ-DNA adduct formation, persistence and tumorigenicity in target organs, young adult male and female CDF1 mice were treated with a single p.o. dose (50 mg/kg) of IQ, and IQ-DNA adducts were isolated and quantitated in liver, lungs, forestomach, small intestine and large intestine over a 24 day period. In the range of 5-50 mg IQ/kg, IQ-DNA adduct formation was related to dose in all organs examined (liver, lungs, stomach, small intestine, large intestine). Total adduct formation (expressed as relative adduct labeling, RAL) 24 h after administration was highest in the liver (6.4-6.9 x 10(-7)) with lower levels, in decreasing order, in the large intestine, small intestine (non-target organs), forestomach and lungs (target organs). In all cases the major adduct, comprising 68-79% of the total, co-chromatographed with standard N-(deoxyguanosin-8-yl)-IQ. Up to three additional IQ-specific adducts could be detected. Except in the liver, adduct levels 24 h after administration of IQ were 2- to 3-fold higher in females than in males. There was no preferential retention of any one of the four adducts 1-24 days after administration of IQ. Beyond day 4 after administration, total adducts in the liver of females were approximately 2-fold higher than those in males, and the rate of removal from female lung was approximately 2-fold faster than that from male lung during the 1-24 day time period. Both these findings correspond to the known sex differences in IQ-induced tumor incidences in these organs. The higher adduct levels in non-target organs (intestines) as compared to target organs (lungs and stomach), combined with the absence of differential persistence of any individual adduct indicates that, in addition to adduct formation and persistence, other factors contribute to the target organ specificity of IQ in CDF1 mice.

Formation and persistence of DNA adducts of 2-amino-3-methylimidazo[4,5-f] quinoline in male Fischer-344 rats.

The mutagenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) is carcinogenic in the Fischer-344 rat, affecting the liver and small and large intestines, as well as several other organs. In male animals the incidences of tumors in the liver, small intestine, and large intestine were reported to be 67.5, 30.0, and 62.5%, respectively. Using 32P-postlabeling assays, the formation and persistence of IQ-DNA adducts in the liver and small and large intestines were studied in male Fischer-344 rats. Young, adult animals were either given a single p.o. dose (5, 25, or 50 mg/kg) of IQ and were killed 24 h later or were given a single p.o. or i.p. dose (50 mg/kg) of IQ and were killed at different time points, from 6 h to 31 days after p.o. treatment and from 6 h to 6 days after i.p. treatment, to follow adduct persistence. Up to five specific adducts could be isolated, and adduct formation was dose related in all three organs. Adduct 1, previously shown to be N-(hydroxyguanosin-8-yl)-IQ, was the major adduct in all cases, comprising up to 78% of the total. After p.o. administration (6-24 h) adduct levels in the liver were 3- to 4-fold higher than after i.p. administration, while levels in the intestines during this time period were independent of the route of administration. At 24 h after p.o. administration total adduct levels in the liver were 13.5-41.4 and 9.2-18.4 times higher than those in the small intestine and large intestine, respectively. Maximum adduct levels were observed between 6 and 24 h after administration, and from 1 to 6 days later, rates of removal from the liver were 7-fold and 2-fold slower, respectively, than from the small and large intestine. Rates of adduct removal from the intestines after i.p. administration were similar to those after p.o. administration. Beyond day 15 adduct levels in all organs constituted less than 12% of those on day 1, and low levels of adducts persisted for up to 31 days. In all cases there was no preferential loss or persistence of any of the adducts. It is concluded that total adduct levels and persistence in target organs may, in part, be related to susceptibility to IQ-induced carcinogenesis in the Fischer-344 rat.

Similar patterns of DNA adduct formation of 2-amino-3-methylimidazo [4,5-f]quinoline in the Fischer 344 rat, CDF1 mouse, cynomolgus monkey and Salmonella typhimurium.

2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is a known liver carcinogen in the Fischer 344 rat, the CDF1 mouse and in the cynomolgus monkey. Using 32P-postlabeling assays, we compared IQ-DNA adduct formation in the liver of IQ-treated Fischer 344 rats, CDF1 mice and cynomolgus monkeys with that in Salmonella typhimurium (strain TA98) incubated with IQ (in the presence of a liver S9 activating system) or N-hydroxy-IQ. Up to five adducts could be detected, the pattern of which was identical in all cases. The major adduct co-chromatographed with standard N-(deoxyguanosin-8-yl)-IQ in all cases and comprised 54.7-82.8% of the total. The four minor adducts were not identified. It is concluded that N-(deoxyguanosin-8-yl)-IQ is the major IQ-DNA adduct under all experimental conditions and that the pattern of N-hydroxy-IQ-DNA adducts is identical to that found in the liver of animals exposed to IQ, and to that found after reacting IQ with DNA in the presence of a liver S9 activating system. Thus, N-hydroxylation of IQ is a critical step in the formation of IQ-DNA adducts.