Individual Differences in the Response of Human ß-Lymphoblastoid Cells to the Cytotoxic, Mutagenic, and DNA-Damaging Effects of a DNA Methylating Agent, N-Methylnitrosourethane.

Metabolic activation of many carcinogens leads to formation of reactive intermediates that form DNA adducts. These adducts are cytotoxic when they interfere with cell division. They can also cause mutations by miscoding during DNA replication. Therefore, an individual's risk of developing cancer will depend on the balance between these processes as well as their ability to repair the DNA damage. Our hypothesis is that variations of genes participating in DNA damage repair and response pathways play significant roles in an individual's risk of developing tobacco-related cancers. To test this hypothesis, 61 human B-lymphocyte cell lines from the International HapMap project were phenotyped for their sensitivity to the cytotoxic and genotoxic properties of a model methylating agent, N-nitroso-N-methylurethane (NMUr). Cell viability was measured using a luciferase-based assay. Repair of the mutagenic and toxic DNA adduct, O6-methylguanine (O6-mG), was monitored by LC-MS/MS analysis. Genotoxic potential of NMUr was assessed employing a flow-cytometry based in vitro mutagenesis assay in the phosphatidylinositol-glycan biosynthesis class-A (PIG-A) gene. A wide distribution of responses to NMUr was observed with no correlation to gender or ethnicity. While the rate of O6-mG repair partially influenced the toxicity of NMUr, it did not appear to be the major factor affecting individual susceptibility to the mutagenic effects of NMUr. Genome-wide analysis identified several novel single nucleotide polymorphisms to be explored in future functional validation studies for a number of the toxicological end points.

Role of aldehydes in the toxic and mutagenic effects of nitrosamines.

α-Hydroxynitrosamine metabolites of nitrosamines decompose to a reactive diazohydroxide and an aldehyde. To test the hypothesis that the aldehydes contribute to the harmful effects of nitrosamines, the toxic and mutagenic activities of three model methylating agents were compared in Chinese hamster ovary cells expressing or not expressing human O6-alkylguanine DNA alkyltransferase (AGT). N-Nitrosomethylurethane (NMUr), acetoxymethylmethylnitrosamine (AMMN), and 4-(methylnitrosamino)-4-acetoxy-1-(3-pyridyl)-1-butanone (NNK-4-OAc) are all activated by ester hydrolysis to methanediazohydroxide. NMUr does not form an aldehyde, whereas AMMN generates formaldehyde, and NNK-4-OAc produces 4-oxo-1-(3-pyridyl)-1-butanone (OPB). Since these compounds were likely to alkylate DNA to different extents, the toxic and mutagenic activities of these compounds were normalized to the levels of the most cytotoxic and mutagenic DNA adduct, O6-mG, to assess if the aldehydes contributed to the toxicological properties of these methylating agents. Levels of 7-mG indicated that the differences in cytotoxic and mutagenic effects of these compounds resulted from differences in their ability to methylate DNA. When normalized against the levels of O6-mG, there was no difference between these three compounds in cells that lacked AGT. However, AMMN and NNK-4-OAc were more toxic than NMUr in cells expressing AGT when normalized against O6-mG levels. In addition, AMMN was more mutagenic than NNK-4-OAc and MNUr in these cells. These findings demonstrate that the aldehyde decomposition products of nitrosamines can contribute to the cytotoxic and/or mutagenic activity of methylating nitrosamines.

Dosing and scheduling influence the antitumor efficacy of a phosphinic peptide inhibitor of matrix metalloproteinases.

The in vivo disposition and antitumor efficacy of a newly developed phosphinic matrix metalloproteinase inhibitor (RXP03) were examined. RXP03 potently inhibits MMP-11, MMP-8 and MMP-13, but not MMP-1 and MMP-7. Twenty-four hours after i.p. injection into mice, most of the RXP03 was recovered intact in plasma, feces (biliary excretion) and tumor tissue. Pharmacokinetic parameters indicated that, after an i.p. dose of 100 microg/day, the plasma concentration of RXP03 over 24 hr remained higher than the Ki values determined for MMP-11, MMP-8 and MMP-13. Efficacy of RXP03 on the growth of primary tumors induced by s.c. injection of C(26) colon carcinoma cells in mice was observed to depend both on RXP03 doses and treatment schedules. Tumor volumes in mice treated for 18 days with 50, 100 and 150 microg/day of RXP03 were decreased compared with control tumor volumes, 100 microg/day being the most effective dose. Treatment at higher dose (600 microg/day) did not significantly reduce the tumor size as compared to control. Short treatments with RXP03 100 microg/day, 3 to 7 days after C(26) inoculation, were more effective on tumor growth than continuous treatment over 18 days. Strikingly, RXP03 treatment started 6 days after the C(26) injection and continued until day 18 led to stimulation of tumor growth, as compared to control. These paradoxical effects, depending on the RXP03 treatment schedule, underline the need to define carefully the spatiotemporal function of each MMP at various stages of tumor growth to achieve optimal therapeutic effects by MMP inhibitor treatment.

Genotoxic methylating agents modulate extracellular signal regulated kinase activity through MEK-dependent, glutathione-, and DNA methylation-independent mechanisms in lung epithelial cells.

Mitogen-activated protein kinases (MAPKs) play a central role in transmitting stress-induced signals stimulated by genotoxic agents. The present study is the first to investigate the mechanisms by which genotoxic alkylating agents modulate MAPKs by directly measuring the effects of methylating agents on MAPK activity, DNA methylation, and intracellular glutathione levels. The effects of acetoxymethylmethylnitrosamine (AMMN), N-nitroso-N-methylurethane (NMUR), and N-methyl-N-nitrosourea (MNU) on these parameters were compared in a fetal rat lung cell line model (MP48). These compounds were chosen because they methylate DNA via a methanediazonium intermediate and, therefore, should induce similar cellular methylation patterns, although they produce different side products upon decomposition. All three compounds stimulated the activation of the stress-activated MAPKs, c-Jun N-terminal kinase, and p38. In contrast to what has been reported for other methylating agents, these compounds also stimulated the activation of extracellular signal regulated kinase (ERK), a MAPK typically activated by mitogenic agents. O6-methylguanine (O6-mG) is widely considered to be the critical toxic lesion induced by methylating agents, including AMMN, NMUR, and MNU, which form DNA adducts through SN1 reactions. O6-mG does not appear to be a key regulator of MAPK activity by these compounds, however. There is no direct relationship between the levels of O6-mG and the levels of MAPK activation, and formation of O6-mG does not appear to be sufficient to stimulate MAPK activation. The present studies also indicate that depletion of glutathione is not required or sufficient to stimulate MAPK activation by the methylating agents investigated here. The use of a pharmacological inhibitor indicates that these methylating agents activate ERK through a signaling pathway that requires the ERK kinase MEK. Altogether, these data indicate that genotoxic methylating agents activate MAPKs through mechanisms that are likely to involve the alkylation of cellular targets other than DNA.

Different genetic alterations in rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens.

Human beings are exposed to a multitude of carcinogens in their environment, and most cancers are considered to be chemically induced. Here we examined differences in genetic alterations in rat forestomach tumors induced by repeated exposure to a genotoxic carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methylnitrosourethane (MNUR), and chronic treatment with a non-genotoxic carcinogen, butylated hydroxyanisole (BHA) or caffeic acid (CA). A total of 132, 6-week-old male F344 rats were employed. Forty rats were treated with MNNG by intragastric administration at a dose of 20 mg/kg body wt once a week for 32 weeks, and 20 rats received 20 p.p.m. MNUR in their drinking water for 48 weeks. Further groups of 20 animals were administered 2% BHA or 2% CA in the diet for 104 weeks. The remaining rats were maintained without any supplement as controls. Multiple forestomach tumors were observed in all rats of the MNNG-, MNUR-, BHA- and CA-treated groups. Histopathologically, MNUR- and CA-treated groups showed almost the same pattern. On polymerase chain reaction-single strand conformation polymorphism analysis, H-ras and p53 gene mutations were observed at high and relatively low frequencies, respectively, in forestomach tumors induced by MNNG and MNUR. Most H-ras gene mutations were G-->A transitions in codons 7 and 12 of exon 1. On the other hand, forestomach tumors due to the non-genotoxic carcinogens, BHA and CA, had almost no mutations of the H-ras and p53 genes. Moreover, relative overexpression of cyclin D1 and p53 was detected in forestomach tumors induced by the genotoxic carcinogens, while their non-genotoxic counterparts had a tendency to show low expression of those molecules. Mutations of the beta-catenin gene were not detected in any group. The present study demonstrates that rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens have different underlying genetic alterations, even if their pathological features are similar.

Alveolar environment influences the metabolic and biophysical properties of exogenous surfactants.

Several factors have been shown to influence the efficacy of exogenous surfactant therapy in the acute respiratory distress syndrome. We investigated the effects of four different alveolar environments (control, saline-lavaged, N-nitroso-N-methylurethane, and hydrochloric acid) on the metabolic and functional properties of two exogenous surfactant preparations: bovine lipid extract surfactant and recombinant surfactant-associated protein (SP) C drug product (rSPC) administered to each of these groups. The main difference between these preparations was the lack of SP-B in the rSPC. Our results demonstrated differences in the large aggregate pool sizes recovered from each of the experimental groups. We also observed differences in SP-A content, surface area cycling characteristics, and biophysical activities of these large aggregate forms after the administration of the two exogenous surfactant preparations. We conclude that the alveolar environment plays a critical role, influencing the overall efficacy of exogenous surfactant therapy. Thus further preclinical studies are warranted to investigate the specific factors within the alveolar environment that lead to the differences observed in this study.

Ventilation strategies affect surfactant aggregate conversion in acute lung injury.

This study evaluated the effects of varying tidal volumes (VT) and positive end-expiratory pressure (PEEP) levels on surfactant aggregate conversion and lung function in an animal model of lung injury induced by N-nitroso-N-methylurethane. Lung-injured adult rabbits were initially ventilated using a VT of 10 ml/kg (VT10), a respiratory rate of 30 breaths/min (RR30), and a PEEP of 3.5 cm H2O. A trace dose of radiolabeled rabbit large surfactant aggregates was instilled after the onset of ventilation, and animals were then ventilated at different ventilator settings for 1 h. Ventilation strategies involving a lower VT (VT5, RR60) resulted in significantly superior oxygenation and lower surfactant aggregate conversion rates than strategies involving a higher VT ([VT10, RR30], [VT15, RR20], p < 0.05). Increasing the PEEP level to 8.0 cm H2O improved oxygenation, but it was sustained only with a low VT (VT5, RR60), and deteriorated with a high VT (VT10, RR30). Varying VT but not PEEP levels resulted in significant changes in surfactant aggregate conversion. We conclude that increased surfactant aggregate conversion resulting from suboptimal ventilation of injured lungs may play an important role in the pathophysiology of ventilation-induced lung dysfunction in acute lung injury.

Inhibition of nitric oxide synthase attenuates NNMU-induced alveolar injury in vivo.

The purpose of this study was to determine if the acute alveolar injury induced by subcutaneous injections of N-nitroso-N-methylurethane (NNMU) in rats is mediated by nitric oxide (NO.). We show that intraperitoneal injections of the NO. synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine significantly attenuate the NNMU-induced alveolar injury as assessed by 1) normalization of the alveolar-arterial O2 difference, 2) attenuation of the lowered phospholipid-to-protein ratio in the crude surfactant pellet (CSP), 3) attenuation of the elevated minimal surface tension of the CSP, and 4) attenuation of polymorphonuclear neutrophilic infiltration into the alveolar space. Injections of N omega-nitro-D-arginine methyl ester, the inactive stereoisoform of L-NAME, did not affect the acute lung injury. Western blot analysis of whole lung homogenates demonstrate an elevated expression of transcriptionally inducible, Ca(2+)-independent NOS (iNOS) in NNMU-injected rats compared with control saline-injected rats. NOS inhibitors did not affect NNMU-induced iNOS expression. These investigations demonstrate that the inhibition of NOS attenuates NNMU-induced acute lung injury, suggesting a role for NO. in the progression of acute respiratory distress syndrome.

DNA single-strand scission in the pyloric mucosa of rat stomach induced by four glandular stomach carcinogens and three other chemicals.

Induction of DNA single-strand scission by four glandular stomach carcinogens and three other chemicals was studied in the pyloric mucosa of rat stomach after gastric intubation. DNA single-strand scission, as was measured by the alkaline elution method, was induced by four glandular stomach carcinogens; N-nitroso N-methylurethane at doses of 1 and 9 mg/kg body wt, 4-nitroquinoline 1-oxide at 20 and 30 mg/kg body wt. N-ethyl-N'-nitro-N-nitrosoguanidine at 30 and 100 mg/kg body wt and N-propyl-N'-nitro-N-nitrosoguanidine at 30 and 100 mg/kg body wt. DNA single-strand scission was also induced dose-dependently by a direct acting mutagen, 1-nitrosoindole-3-acetonitrile at doses of 100, 500 and 800 mg/kg body wt. Omeprazole, a proton pump inhibitor, was equivocal in its effect in this assay at 30-500 mg/kg body wt: induction was statistically significant by Cochran-Armitage binomial trend test. Loxtidine, an H2-receptor antagonist, did not induce DNA single-strand breaks in the pyloric mucosa at a dose of 400 mg/kg body wt. The present results together with previous information suggest that DNA single-strand scission is a good marker for tumor-initiating activity in rat stomach mucosa.

Relationship between the development of pulmonary fibrosis and lung tumors in Syrian golden hamsters induced by N-methyl-N-nitrosourethane.

To cast light on the relationship between the development of pulmonary fibrosis and lung cancer, female Syrian golden hamsters were given 5 sc injections of 0.6 mg/animal of N-methyl-N-nitrosourethane (MNUR) at 2-wk intervals and then maintained without any treatment for 26 wk. Bronchiolo-alveolar cell tumors and hyperplasias, which were recognized from week 4 after termination of treatment, were each subdivided morphologically into 3 types. Bronchiolo-alveolar cell tumors included papillary tumors consisting of basophilic cells, papillary tumors consisting of clear cells, and papillary/solid tumors consisting of pleomorphic cells, with papillary tumors consisting of basophilic cells predominating. Bronchiolo-alveolar cell hyperplasias encountered were glandular metaplasia, simple hyperplasia, and papillary hyperplasia. Glandular metaplasia was the most common of the hyperplastic lesions. To some extent, all the proliferative lesions were associated with inflammatory cell infiltration, but this varied greatly. The rate for papillary tumors consisting of basophilic cells with connective tissue proliferation was 35%. Among the hyperplastic lesions, the cell proliferative activity of papillary hyperplasia (12.5%) was significantly higher than in other hyperplastic lesions, suggesting that this lesion might be a preneoplastic change. None of the lung proliferative lesions showed any unequivocal immunoreactivity for the p53 protein. The present study suggests that most lung tumors may develop from pulmonary inflammatory lesions induced by MNUR, but the possibility that DNA injuries to the respiratory epithelial cells by MNUR may cause lung tumors cannot be precluded.

Experimental induction of pulmonary fibrosis in Syrian golden hamsters by N-methyl-N-nitrosourethane.

A range finding study for experimental induction of pulmonary fibrosis in which female Syrian golden hamsters received five subcutaneous injections of 0.8, 0.6, 0.4, 0.2 or 0 mg of N-methyl-N-nitrosourethane (MNUR) once a week or once per two weeks revealed most of the animals of the 0.8 mg group to die of acute pulmonary injury due to MNUR while typical interstitial pneumonia was induced in the 0.6 mg group. Based on these results hamsters were given five subcutaneous injections of 0.6 mg/animal of MNUR once per two weeks and then reared without any treatment for 12 weeks. Marked interstitial edema and intraalveolar infiltration of macrophages due to alveolar capillary damage were seen in treated animals at week 1, and secondary diffuse fibrotic thickening of the alveolar septa, as evidenced by increased type III collagen demonstrated immunohistochemically, was marked thereafter. The content of hydroxyproline in the lung was significantly increased from week 4. The present study indicates that lung injuries attributable to primary damage of alveolar capillaries progress to diffuse alveolar fibrosis in hamsters treated with MNUR, suggesting that this animal model might be of advantage for pathogenetic analysis of the relationship between pulmonary fibrosis and lung cancer development.

Altered surfactant function and metabolism in rabbits with acute lung injury.

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.

A kinetic model of the cell culture cytotoxicity of metabolically activated agents: N-methyl-N-(acetoxymethyl)nitrosamine, methylnitrosourethane, and (methylazoxy)methanol acetate.

The activity of three metabolically activated methylating agents, N-methyl-N-(acetoxymethyl)nitrosamine (DMN-OAc), methylnitrosourethane (MNUT), and (methylazoxy)methanol acetate (MAM-Ac), were determined in cell culture by using a P388 cell growth rate inhibition assay. Experiments were conducted with normal P388 cells in Fischer's medium and under conditions in which the esterase-mediated activation was modified by pretreating cells with the irreversible esterase inhibitor paraoxon and by adding acetylcholinesterase to the medium. Inhibition of intracellular esterase had a much greater effect on activity than did addition of enzyme to medium. These experiments provided data that were used to assess the utility of kinetic models as a means to gain a more detailed understanding of the cytotoxicity process in cell culture. Growth rate inhibition was related to the amount of intracellular alkylation resulting from formation of metabolic intermediates and their subsequent chemical reaction to form methyldiazonium ion and methylation products by using kinetic rate laws and measured rate constants. The model is applicable to systems that form unstable metabolites that can, in part, partition between the cell volume and incubation medium. When growth rate inhibition effects were related to cumulative intracellular alkylation [P], the ED50 values were the same for all three agents and for three previously reported chemically activated methylating agents, N-methyl-N-nitrosourea, streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine, which are also thought to act through the methyldiazonium ion. This observation is consistent with a growth rate inhibition effect of the methyldiazonium ion that reflects the intrinsic activity of this species that is independent of the precursor molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of non-genotoxic carcinogens in the BALB/c 3T3 cell transformation/mutation assay system.

We have employed BALB/c 3T3 cells in a simultaneous cell transformation and mutation assay protocol to see whether both genotoxic and non-genotoxic carcinogens can be identified. Three known mutagenic animal carcinogens tested positive for transforming activity: dimethylnitrosamine, methylcholanthrene and methylnitrosourea. In addition, we tested three non-mutagenic compounds--two human carcinogens, benzene and diethylstilbestrol, and the possible human carcinogen dichlorodiphenyltrichloroethane; all three displayed transforming activity. These results add to existing data supporting the validity of the BALB/c 3T3 cell transformation system as a reliable short-term in vitro test for carcinogens in general, and for non-genotoxic carcinogens in particular.

Comparison of the acute toxicity of N-nitrosocimetidine with three structurally related carcinogens in the rat.

The acute toxicity of N-nitrosocimetidine, the nitrosated derivative of the histamine H2-receptor blocking agent cimetidine, was compared with the toxicities of three structurally related nitroso compounds known to be potent carcinogens, namely N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, and N-methyl-N-nitrosourethane, and also with the parent drug cimetidine. The acute toxicity of each compound was investigated in 6-week-old female Fischer-344 rats by estimating the median lethal doses via three different routes of administration, and by assessing the sequence of histopathological alterations induced. According to median lethalities, all three known carcinogens were substantially more toxic than nitrosocimetidine whether administered by the intravenous, intraperitoneal, or oral routes. The widest margin of difference was represented by orally administered N-methyl-N-nitrosourea, the median lethal dose being 59 times greater than oral N-nitrosocimetidine. By this method, the acute toxicities of N-nitrosocimetidine and the parent drug cimetidine were virtually identical for each of the three routes of administration. Sequential histological assessment indicated that the three known carcinogens induced specific pathological alterations mainly in organs which were also known to be targets for their carcinogenic activity. In contrast, no tissue lesions of a specific nature were associated with N-nitrosocimetidine or cimetidine in this study. The comparable results with N-nitrosocimetidine and the parent drug provide biological support for previously obtained biochemical data which suggested that N-nitrosocimetidine is rapidly denitrosated to cimetidine in the rat.

Normal surface properties of phosphatidylglycerol-deficient surfactant from dog after acute lung injury.

Lung surfactant was isolated from bronchoalveolar lavage of dogs during the late phase of recovery (15 days) from acute alveolar injury induced by subcutaneous injection of N-nitroso-N-methylurethane. This surfactant was compared with surfactant from control dogs in terms of in vitro surface properties, phospholipid composition and protein content, and those of its subfractions. Phospholipid composition and protein content were similar in the two groups, except that phosphatidylglycerol (PG) was markedly reduced and phosphatidylinositol (PI) was increased in the experimental group. In both, isopycnic densities of their subfractions in continuous sucrose density gradient were identical. The time course of surfactant adsorption was similar in both groups. Minimum surface tension (gamma min) was 4.1 +/- 1.5 dynes/cm in the experimental dogs and 3.8 +/- 1.3 dynes/cm in the controls. Surface compressibility (SC), stability index (SI), and dynamic respreadability (DR) of the surfactants from the two groups were nearly identical. When compared to an artificial surfactant composed of dipalmitoyl phosphatidylcholine (DPPC) and PG in 9:1 molar ratio a mixture of DPPC-PI 9:1 prepared identically showed similar gamma min, SC, SI, and DR, and a much higher surface adsorption rate. These results suggest that PG is not essential for normal in vitro surfactant function and that its role may be assumed by PI.

Enhancement of N-bis(2-hydroxypropyl)nitrosamine-initiated lung tumor development in rats by bleomycin and N-methyl-N-nitrosourethane.

Potential modification of N-bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung carcinogenesis by bleomycin and N-methyl-N-nitrosourethane (MNUT) was investigated in male F344 rats. Rats were given 0.2% DHPN for 1 week followed by a weekly i.p. injection of either bleomycin at 2.0 mg/kg body wt or of MNUT at a dose of 1.0 mg/kg body wt for 35 weeks. Animals given DHPN followed by solvent administration alone, either saline or 0.02% ethanol and animals given bleomycin or MNUT without DHPN pretreatment were served as controls. Bleomycin treatment increased the incidence of cancer, whereas hyperplasia, adenoma and cancer of the lung were all significantly elevated after MNUT administration. MNUT alone induced low incidences of lung tumors. Bleomycin also increased the incidence of papillary or nodular hyperplasia of the urinary bladder while it inhibited the development of thyroid tumors. MNUT, however, did not affect tumor development in other organs.

Diphosphatidylglycerol in experimental acute alveolar injury in the dog.

Acute alveolar injury closely resembling that seen in humans was induced in dogs by subcutaneous injection of N-nitroso-N-methylurethane. Necrosis of alveolar epithelial cells was observed during early injury. Proliferation of immature epithelial cells which began during early injury and became massive after peak injury was followed by their differentiation to mature type II cells during recovery. Quantities of diphosphatidylglycerol (DPG) and of phosphatidylglycerol (PG) in alveolar lavage and in post-lavage lung tissue were measured. An increase in tissue DPG coincided with a sharp decrease in tissue and lavage PG during early injury. DPG was not detectable in the lavage. During late recovery, tissue DPG increased threefold over controls. This increase was accompanied by persistence of a 50% decrease in tissue PG and 83% decrease in lavage PG. Biosynthesis of DPG and PG in isolated lung mitochondria demonstrated that DPG was formed from PG in the presence of CDP-diglyceride. These findings suggest that the low level of PG in the surfactant complex during acute alveolar injury is due to increased turnover of PG to DPG in the lung.

Carcinogenesis by combinations of N-nitroso compounds in rats.

Five N-nitroso compounds were administered individually or in various combinations to groups of 20 female F344 rats to examine possible additive or synergistic effects on mortality rate and the induction of tumours. Combination of the oesophageal carcinogen dinitroso-2,6-dimethylpiperazine with nitrosodiethylamine, which also induces oesophageal tumours, or with nitrosoethylmethylamine, which induces liver tumours, did not appear to increase tumour incidence. Similarly, combination of nitrosodiethylamine with the bladder carcinogen nitrosomethyldodecylamine did not increase the formation of either bladder or oesophageal tumours compared with the effects of either compound alone, although an additive effect was suggested by a higher incidence of liver tumours after the combined treatment than after treatment with either compound alone. Nitrosomethylurethane, which induces tumours of the forestomach in rats, appeared to enhance the effectiveness of nitrosoethylmethylamine and dinitroso-2,6-dimethylpiperazine in inducing tumours of the oesophagus, and increased the rate of death. Nitrosomethylurethane given, at a somewhat higher dose, in combination with both of these compounds increased the incidence of oesophageal tumours even when the treatment lasted only 4 wk. This evidence suggests that additive or synergistic effects of combinations of N-nitroso compounds at low dose rates can be demonstrated in small groups of rats.

Enhancement by bracken of induction of tumours of the upper alimentary tract by N-propyl-N-nitrosourethan.

The effect of bracken on the induction of tumours of the upper alimentary tract by N-propyl-N-nitrosourethan (PNU) was studied in 7-week-old ACI rats. Group I received a solution of 400 pts/10(6) of PNU in their drinking water for 6 weeks; Groups II and III were given PNU as in Group I, and then from 1 week later were fed on diets containing 5 and 30% bracken, respectively, for 33 weeks; Groups IV and V were fed on diets containing 5 and 30% bracken, respectively, for 33 weeks, from 14 weeks after birth. A control group was given basal diet and water only. The experiment was terminated after 40 weeks. The induction of tumours of the upper alimentary tract by PNU was enhanced by bracken diet; i.e. the incidence of pharyngeal tumours in male rats was significantly higher (P less than 0.025) in Group II (10/13) than in Group I (3/13). The incidence and multiplicity of oesophageal tumours in female rats were also higher in Group III than in Group I (P less than 0.025 for incidence; P less than 0.05 for multiplicity). Histologically, the oesophageal tumours in female rats in Groups II and III were not only papillomas but also squamous-cell carcinomas, whereas those in females of Group I were all papillomas. Furthermore, the incidence of tumours of the forestomach in female rats was also higher (P less than 0.05) in Group II (11/13) than in Group I (4/12).