Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system.

Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT-/-) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT-/- rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

Nitric oxide participates in early events associated with NNMU-induced acute lung injury in rats.

In this study, the biochemical mechanisms by which N-nitroso-N-methylurethane (NNMU) induces acute lung injury are examined. Polymorphonuclear neutrophil infiltration into the lungs first appears in the bronchoalveolar lavage (BAL) fluid 24 h after NNMU injection (10.58 +/- 3.00% of total cells; P < 0.05 vs. control animals). However, NNMU-induced elevation of the alveolar-arterial O2 difference requires 72 h to develop. Daily intraperitoneal injections of the inducible nitric oxide (. NO) synthase (iNOS)-selective inhibitor aminoguanidine (AG) initiated 24 h after NNMU administration improve the survival of NNMU-treated animals. However, AG administration initiated 48 or 72 h after NNMU injection does not significantly improve the survival of NNMU-treated animals. These results suggest that. NO participates in events that occur early in NNMU-induced acute lung injury. BAL cells isolated from rats 24 and 48 h after NNMU injection produce elevated. NO and express iNOS during a 24-h ex vivo culture. AG attenuates. NO production but does not affect iNOS expression, whereas actinomycin D prevents iNOS expression and attenuates. NO production by BAL cells during this ex vivo culture. These results suggest that NNMU-derived BAL cells can stimulate iNOS expression and. NO production during culture. In 48-h NNMU-exposed rats, iNOS expression is elevated in homogenates of whole lavaged lungs but not in BAL cells derived from the same lung. These findings suggest that the pathogenic mechanism by which NNMU induces acute lung injury involves BAL cell stimulation of iNOS expression and. NO production in lung tissue.

Effect of N-nitroso-N-methylurethane on gas exchange, lung compliance, and surfactant function of rabbits.

OBJECTIVES: To define the effect of N-nitroso-N-methyl-urethane (NNNMU) on pulmonary gas exchange, compliance and the biochemical and functional properties of the lung surfactant system. DESIGN: Four days after inducing lung injury, gas exchange and pulmonary compliance were studied and a bronchoalveolar lavage was taken. SETTING: Experimental laboratory of a university department of medicine, division of pulmonary and critical care medicine. ANIMALS: Ten rabbits after they had received an injection of NNNMU and five control animals. INTERVENTIONS: Controlled mechanical ventilation and bronchoalveolar lavage. MEASUREMENTS AND RESULTS: Measurements of gas exchange (using the multiple inert gas elimination technique), hemodynamics and pulmonary compliance were performed during ventilatory and hemodynamic steady state. A bronchoalveolar lavage (BAL) was taken after sacrificing the animal. BAL samples were processed for cell count and biochemical and functional surfactant analysis. Animals injected with NNNMU developed mild, but significant reduction in PaO2, while maintaining eucapnia during spontaneous air breathing. V/Q distributions and arterial blood gases were similar in all animals when ventilated mechanically with a fixed tidal volume. Compliance of the lung and phospholipid levels in lavage of NNNMU animals was significantly lower than in control animals (CON). Function of surfactant recovered from animals receiving NNNMU was decreased significantly where compared to CON. Thus, NNNMU resulted in a lowered lavage surfactant phospholipid content, impaired surfactant function, decreased compliance and hypoxemia during spontaneous ventilation. However, gas exchange was similar to that of control animals during mechanical ventilation. CONCLUSION: We conclude that NNNMU-induced gas exchange abnormalities present after 4 days are mild and are reversed by fixed volume mechanical ventilation despite marked alteration in surfactant function and lung compliance. These observations further define properties of a lung injury model that is of value in the study of surfactant replacement.

Alveolar surfactant aggregate conversion in ventilated normal and injured rabbits.

Alveolar surfactant can be separated into two subtypes; large aggregates and small aggregates. Large aggregates represent the surface active form of surfactant and are the metabolic precursors of small aggregates. Previous studies examined the mechanism by which large aggregates are converted into small aggregates in vitro. We used intratracheal injection of radiolabeled large aggregates in rabbits to probe the aggregate conversion in vivo. After this injection, animals were mechanically ventilated for 60 min. After the animals were killed, the lungs were lavaged, and the percentage of radiolabel present in the small aggregate fraction was determined. Our results showed that ventilation resulted in aggregate conversion and that increases in tidal volume, but not in respiratory rate, correlated with increased conversion. Aggregate conversion in rabbits with acute lung injury correlated significantly with severity of injury. We conclude that a change in surface area (i.e., respiration) is necessary for aggregate conversion in vivo and that the ventilation strategy can affect this conversion. Furthermore, increased aggregate conversion in injured lungs might contribute to increased small-to-large aggregate ratios in these lungs compared with normal lungs.

Lack of ultraviolet mutagenesis in radiation-resistant bacteria.

Ultraviolet (UV) radiation did not induce rifampicin-resistant mutants in populations of the taxonomically-related radiation-resistant bacteria Deinococcus radiodurans, D. radiopugnans, D. radiophilus and D. proteolyticus, although such mutants arose spontaneously at a low frequency and at a high frequency after treatment of cultures with N-nitroso compounds. The radiation-resistant bacteria Arthrobacter radiotolerans and P-30-A were also UV-immutable whereas the more radiation-sensitive Pseudomonas radiora was UV-mutable. We conclude that the radiation-resistant bacteria repair UV-induced DNA damage accurately and lack an error-prone pathway for the repair of such damage.

Supermutagenicity of ethylnitrosourea in the mouse spot test: comparisons with methylnitrosourea and ethylnitrosourethane.

The effects of the 3 related compounds ethylnitrosourea (ENU), methylnitrosourea (MNU), and ethylnitrosourethane (NEC) were studied in the mouse spot test. ENU induces a large number of recessive spots (RS) and, due to its low toxicity, can be applied at relatively high doses. This combination of properties makes it the most efficient spot-test mutagen, as shown in a comparison with 16 other chemicals, even though, on the basis of molarity, it is not the most potent one. The ENU mutation frequency in cells at risk, calculated per locus, per unit of applied dose, is roughly similar for melanocyte precursors (in the spot test) and spermatogonial stem cells (in the specific-locus test). MNU which, due to its high embryotoxicity, could be tested only at a low dose, is clearly mutagenic, and dose extrapolations indicate it to be more potent than ENU. NEC, though it could be tested at higher molarities than ENU, is only weakly mutagenic. The spot test, in addition to mutational data, also yields information on cytotoxicity (white midventral spots), embryotoxicity, and teratogenicity. The toxicity and teratogenicity findings parallel earlier results in the rat. For all endpoints studied. ENU is more effective than NEC. Relative to MNU, ENU is less toxic, less teratogenic, and less mutagenic in the spot test; but it is much more carcinogenic (transplacentally) and more mutagenic in spermatogonia. We propose that MNU is more effective in inducing gross chromosomal damage than is ENU, while ENU induces relatively more gene mutations. The spot test scores both types of mutational damage, while mostly the latter type is recovered from spermatogonia.

Lysolecithin acyltransferase and alveolar phosphatidylcholine palmitate in experimental acute alveolar injury in the dog lung.

Lysolecithin acyltransferase (EC 2.3.1.23) activities in lung homogenates and in subcellular fractions, and fatty acid composition of phosphatidylcholine (PC) in lung lavage were studied in dogs with acute alveolar injury induced by N-nitroso-N-methylurethane. The specific activity in the microsomal fraction was 10 and 3 times higher than those of homogenate and mitochondrial fractions, respectively. Both the lysolecithin acyltransferase activities and the proportions of palmitate in alveolar lavage PC increased during the early phase of injury (days 2-4), and decreased during peak injury (days 6-8). Such correlation was not found during the recovery period (day 15). During recovery, specific and total activities of the enzyme were nearly 2- and 3-fold, respectively, those of controls. Nevertheless, the palmitate proportions in PC were normal, indicating that the increased enzyme activity in vitro was not reflected in increased PC palmitate during recovery. This finding indicates that the enzyme activity per cell was normal during recovery and suggests that the increase in specific and total activities is due to massive regeneration of type II cells and that the enzyme is localized mainly in these cells. The decrease in the proportion of palmitate in lavage PC during peak injury may lead to abnormality of surfactant function.

Correlations of DNA damage and repair with nuclear and chromosomal damage in HeLa cells caused by methylnitrosamides.

N-Methyl-N-nitrosourethan induces breaks or alkali-labile sites in cellular DNA, many if not all of which are repaired rapidly. Other DNA lesions are repaired by an excision process. Hydroxyurea and 1-beta-D-arabinofuranosylcytosine cause an accumulation of DNA breaks after N-methyl-N-nitrosourethan treatment, probably by inhibiting the DNA-synthetic (but not the nucleolytic) stage of excision repair. Chromosome damage (fragmentation or attenuation of interphase chromosomes and decondensation and radial rearrangement of metaphase chromosomes) is present soon after treatment with N-methyl-N-nitrosourethan and is not reversed during further incubation. It is apparently associated with the longer-lived DNA lesions, probably those which are removed by excision, and is enhanced by incubation with hydroxyurea and 1-beta-D-arabinofuranosylcytosine. N-Methyl-N-nitrosourethan also inhibits cellular protein and the loss of nucleolar structure. N-Methyl-N-nitrosourea is less potent than N-methyl-N-nitrosourethan in causing DNA or chromosome damage and is less cytotoxic.

Pulmonary microvascular and alveolar epithelial permeability characteristics in N-nitroso-N-methylurethane-injected dogs.

The subcutaneous injection of 5 to 6 mg/kg of body weight of N-nitroso-N-methylurethane (NNNMU) has been reported to cause acute alveolar injury in animals. To determine the permeability characteristics of the alveolar epithelium, we employed the in vivo saline-filled dog lung model and determined the time to 50 percent equilibration in minutes of a specific tracer in the blood and the lung model and determined the time to 50 percent equilibration in minutes of a specific tracer in the blood and the lung liquid (T 1/2) for endogenous serum albumin (MW 69,000 daltons, molecular radius 35 A) and exogenously administered 500,000 MW polydispersed dextrans (molecular radius 200 A). Compared to control animals, T1/2 decreased (permeability increased) in NNNMU-injected dogs from 3,500 +/- 100 to 682 +/- 160 minutes for albumin and from 20,000 +/- 250 to 2,790 +/- 750 minutes for 500,000 MW dextran (P less than 0.001). To determine the permeability characteristics of the pulmonary microvasculature, we employed the right lymph duct cannulation dog model and measured lymph flow/30 minutes, lymph albumin and dextran concentration, and lymph/plasma albumin and dextran ratios in control and NNNMU-injected dogs. Compared to control animals, lymph flow was significantly greater in NNNMU dogs, 2.07 +/- 1.1 vs .71 +/- .50 ml/30 minutes (P less than 0.01), respectively. We conclude that NNNMU injection increases permeability in both the alveolar epithelium and the pulmonary microvasculature.

Concentration-dependent mutation by alkylating agents in human lymphoblasts and Salmonella typhimurium: N-methyl-N-nitrosourethane and beta-propiolactone.

The toxic and mutagenic effects of the alkylating agents N-methyl-N-nitrosourethane (MNUT) and beta-propiolactone (BPL) were quantitatively measured in human lymphoblasts and Salmonella typhimurium. Forward mutation to 6-thioguanine resistance was measured in the human lymphoblasts, and forward mutation to 8-azaguanine resistance was measured in the bacterial cells after equigenerational (1.5 doubling times) exposures. In both systems, the induced mutant fraction rose linearly as a function of concentration for BPL and was biphasic for MNUT. The responses of the two assay systems to eight alkylating agents were compared. The exposure of the cells to each alkylating agent was calculated as exposure concentration multiplied by the time of exposure, and allowance was made for the decomposition of the alkylating agents during exposure (integral exposure). Human cells were 2.5--13 times more sensitive than was S. typhimurium to the alkylating agents methyl methanesulfonate, ethyl methanesulfonate, propyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, methylnitrosourea, and MNUT. S. typhimurium cells were three times more sensitive to butyl methanesulfonate and 25 times more sensitive to BPL than were human cells.

Diaminobenzidine reactions in control and treated Amoeba proteus.

Cytochrome oxidase activity was demonstrated in Amoeba proteus by diaminobenzidine (DAB) cytochemistry. Deposition of the reaction product occurred on the inner mitochondrial membranes and the cristae. The reaction was abolished by cyanide incubations. Positive reactions were produced with both unfixed and fixed cells: although staining potential was destroyed by any prefixatives which included glutaraldehyde. Cells prefixed with 4% formaldehyde, to raise structural preservation, retained staining ability. Amoebae subjected to prolonged anaerobiosis or to treatment with the carcinogen N-methyl-N-nitrosourethane (MNU) displayed a reduction in DAB reactivity. A positive reaction was only produced in incubations of unfixed cells and even in these the intensity of cristal staining was depleted. The possible use of DAB reactions where lesions in mitochondrial functioning have occurred is considered.

Evidence of DNA repair in the guinea pig pancreas, in vivo and in vitro, following exposure to N-methyl-N-nitrosourethane.

The nature of DNA damage induced by N-methyl-N-nitrosourethane (NMUT) in the guinea pig pancreas, both in vitro and in vivo, and subsequent repair was investigated by alkaline sucrose density gradient analysis, using a non-radioactive fluorimetric procedure for DNA determination in gradient fractions. In vitro exposure of pancreatic slices to 20 mM NMUT for 30 min damaged DNA to less than 2.24 . 10(6) dalton fragments. However, incubation of NMUT-treated slices for 3 h in a fresh medium resulted in the repair of most of DNA damage, as indicated by the conversion of low molecular weight DNA fragments into heavy DNA of molecular weight comparable to DNA from control slices. Additionally, a single administration of NMUT (30 mg/kg, i.p.) to guinea pigs induced extensive DNA damage, to less than 2.24 . 10(6) dalton fragments in the pancreas within 4 h; similar DNA damage was observed in the liver. However, in the pancreas and liver of guinea pigs sacrificed at increasing intervals after NMUT administration, there was a gradual conversion of shortened DNA fragments to heavy high molecular weight DNA, indicating repair of DNA damage. It appears that most of DNA damage in the pancreas and liver was repaired by 14 and 7 days, respectively, following NMUT administration.

Effect of chemical carcinogens on pancreatic DNA synthesis in vivo.

The effect of dimethylnitrosamine (DMN), ethionine, streptozotocin, N-methyl-N-nitrosourethan (MNUT), N-bis(2-hydroxypropyl)nitrosamine (DHPN), azaserine, and 4-hydorxyaminoquinoline 1-oxide (4-HAQO) on DNA synthesis after partial pancreatectomy was studied. Its results indicates that DNA synthesis of the residual pancreas occurred and reached the maximum value at 3 days and returned to the control value 12 days after the operation. DNA synthesis, which was inhibited 96.7% by hydroxyurea 3 days after the operation, indicated that semi-conservative DNA synthesis had occurred in the residual pancreas. DMN and ethionine, non-pancreatic carcinogens, did not inhibit while DHPN, MNUT, azaserine, and 4-HAQO, pancreatic non-endocrine carcinogens, inhibited DNA synthesis in the rat pancreas by 57.9, 76.4, 71.7, and 82.1%, respectively. The effect of streptozotocin, pancreatic endocrine carcinogen, on DNA synthesis was not clear from the present experiment. Further effect on the inhibition of DNA synthesis by these carcinogens was obtained by dose-response studies and its results indicated that there was a correlation between pancreatic carcinogens and the inhibition of DNA synthesis after partial pancreatectomy in rats.

Effects of N-methyl-N-nitrosourethane on DNA synthesis in the guinea pig pancreas.

The effects of N-methyl-N-nitrosourethane (NMUT) on pancreatic DNA synthesis were investigated at sequential intervals following gavage of Hartley guinea pigs with a single dose of 30 mg/kg. There was a highly significant stimulation of DNA synthesis, as evidenced by increased incorporation of [3H] methyl-thymidine ([3H]TdR), throughout the whole pancreas and particularly in the duodenal segment, at 4 h following NMUT administration, thereafter, DNA synthesis declined sharply up to 24 h, and then recovered gradually to control levels from 24--96 h. DNA synthesis stimulated by NMUT was suppressed by hydroxyurea (HU), and hence is likely to represent replicative, rather than repair, synthesis.

Kinetics of DNA repair synthesis in guinea-pig pancreatic slices following in vitro exposure to N-methyl-N-nitrosourethane.

In vitro exposure of guinea-pig pancreatic slices to NMUT resulted in an increase in hydroxyurea-insensitive 3H-TdR incorporation into DNA; this represents DNA repair synthesis following NMUT-induced DNA damage. The kinetics of this hydroxyurea-insensitive 3H-TdR incorporation suggest that the NMUT-induced DNA damage is largely repaired within 2 hours.

DNA repair synthesis in guinea pig pancreatic slices following in vitro exposure to N-nitrosomethylurethane.

The incorporation of thymidine into DNA in the presence of hydroxyurea (HU) by guinea pig pancreatic slices following exposure to N-nitrosomethylurethane (NMUT) was used to follow DNA repair synthesis. HU was used to suppress normal replicative DNA synthesis. Slices from the duodenal segment of the pancreas were exposed for periods of 15 to 90 min to NMUT at concentrations of 2 to 20 mM, then incubated in tritiated thymidine ([H3]-TdR) free of carcinogen, and radioactivity in DNA was determined. NMUT induced a a dose- and time-dependent increase in HU-insensitive thymidine incorporation. This stimulated incorporation, which could be attributed to repair synthesis, occurred immediately following the treatment and was largely complete within 3 h.