Importance of cerebral perfusion pressure management using cerebrospinal drainage in severe traumatic brain injury.

OBJECTIVE: To evaluate hemodynamics in patients with severe traumatic brain injury (TBI) after cerebral perfusion pressure (CPP) management using cerebrospinal fluid (CSF) drainage. METHODS: Twenty-six patients with TBI (Glasgow Coma Score = 8 or less) were investigated. Mean arterial blood pressure, CPP, cardiac index (CI), systemic vascular resistance index (SVRI), and central venous pressure were measured. The patients were divided into 2 groups after craniotomy: the intraparenchymal ICP (IP-ICP) monitoring group (n = 14) and ventricular ICP (V-ICP) monitoring group (n = 12). Patient hemodynamics were investigated on the second hospital day to identify differences. Measurements indicated a target CPP above 70 mmHg and a central venous pressure of 8 10 mmHg in both groups. Mannitol administration (IP-ICP group) or CSF drainage (V-ICP group) was performed whenever the CPP remained below 70 mmHg. RESULTS: High SVRI and low CI (p < 0.05) were observed in the IP-ICP group. The V-ICP group exhibited a reduction in the total fluid infusion volume of crystalloid (p < 0.01) and a reduction in the frequency of hypotensive episodes after the mannitol infusion. CONCLUSIONS: CPP management using CSF drainage decreases the total infusion volume of crystalloid and may reduce the risk of aggravated brain edema after excess fluid resuscitation.

High susceptibility of p53(+/-) knockout mice in N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder carcinogenesis and lack of frequent mutation in residual allele.

The loss of p53 functions is considered to compromise the growth-suppression machinery of the cell and facilitate neoplastic change. In humans, genetic alteration in the p53 gene is one of the most frequently observed molecular changes in tumors, including urinary bladder carcinomas. We have investigated the susceptibility of heterozygote p53 knockout mice to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in terms of urinary bladder tumor induction. Both p53(+/-) knockout mice and C57BL/6 original parent strain were administered 0, 0.002, 0.004, 0.0075 and 0.025% BBN in the drinking water for 20 weeks. As compared with the C57BL/6 strain, greater lesion yields were observed in knockout mice after 20 weeks of treatment. Transitional cell carcinomas were found in 9 (75%) and 12 (100%) of each 12 mice of the 0.0075 and 0.025% BBN treatment groups, respectively, whereas only 1 (11%) and 6 (67%) of each 9 of the C57BL/6 mice demonstrated tumors. Preneoplastic lesions (dysplasia) were also observed more frequently in the lower dose groups in the knockout mice than C57BL/6 mice. PCR single-strand conformation polymorphism analysis followed by DNA direct sequencing of the p53 gene (exons 5-8) extracted from bladder tumors demonstrated mutations in 3 of 11 (27.3%; exon 7) and 8 of 29 (27.6%; exons 5-8) tumors in C57BL/6 and knockout mice, respectively. There was no significant difference in the mutation rates at the residual p53 gene between the two cases. All mutations observed in knockout mice were restricted to the normal allele, and none were present in the gene-targeted null allele. In a separate experiment, 5-bromo-2'-deoxyuridine labeling indices after treatment with BBN for 2 or 4 weeks were significantly higher in knockout mice than wild-type mice. Measurement of the urinary concentration of N-butyl-N-(3-carboxypropyl)nitrosamine, a proximate carcinogenic metabolite, revealed no significant differences between knockout and original parent strain after administration of 0.0075% BBN in the drinking water for 4 weeks. In conclusion, knockout mice are distinctly more sensitive to urinary bladder carcinogenesis induced by BBN than their original parent strain, as evidenced by elevated DNA synthesis during carcinogen administration and an increased tumor yield. The high susceptibility of p53 knockout mice appeared to be related to the high level of cell proliferation rather than that of N-butyl-N-(3-carboxypropyl)nitrosamine in the urine or that of mutations at the p53 gene.

Radioiodinated 2'-iododiazepam: a potential imaging agent for SPECT investigations of benzodiazepine receptors.

2'-Iododiazepam (2'-IDZ) is the diazepam analogue iodinated at the 2'-position of C-5 phenyl ring which was synthesized and evaluated as a potential radiopharmaceutical for investigating brain benzodiazepine receptors by SPECT. The 125I-2'-iododiazepam was synthesized by halogen exchange reaction and purified by HPLC. In vitro competitive binding studies with 3H-diazepam, using rat cortical synaptosomal membranes, showed that the affinity of 2'-IDZ for benzodiazepam receptors was higher than that in diazepam and flumazenil (RO15-1788). Biodistribution studies in mice showed that the brain uptake of 2'-iododiazepam was rapid and profound, and in the brain higher accumulation was found in the cortex than in other regions. Furthermore, the cortical uptake was displaced by benzodiazepinergic compounds. In vivo uptake was assessed by autoradiographic studies. Thus, 2'-iododiazepam bound to benzodiazepine receptors in vivo and therefore holds great potential for in vivo benzodiazepine receptor studies.

Cancer chemopreventive agents, labdane diterpenoids from the stem bark of Thuja standishii (Gord.) Carr.

Seven labdane-type diterpenoids from the stem bark of Thuja standishii (Gord.) Carr. (Cupressaceae) and their analogues showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among these compounds, 15,16-bisnor-13-oxolabda-8(17), 11E-dien-19-oic acid was revealed to have the strongest inhibitory effect on the EBV-EA activation, being stronger than that of beta-carotene which has been intensively studied in cancer prevention using animal models. 15,16-bisnor-13-Oxolabda-8(17), 11E-dien-19-oic acid was also found to exhibit the excellent anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

DNA-adduct formation in the forestomach of rats treated with 3-tert-butyl-4-hydroxyanisole and its metabolites as assessed by an enzymatic 32P-postlabeling method.

Formation of DNA-adducts by 3-BHA or its metabolites, i.e., tert-butyl-1,4-benzoquinone (TBQ) and 5-methoxy-3-tert-butyl-1,2-benzoquinone (3-TBOQ), as well as DNA-adduct formation by 4-nitroquinoline-N-oxide (4NQO), in rat forestomach were examined by an enzymatic 32P-postlabeling assay. Four DNA-adducts were clearly detected in the forestomach after treatment of rats with 4NQO. The sensitivity was 1.9 certain adducts per 10(8) normal nucleotides. On the contrary, no DNA adducts were detected in the forestomach of rats given either a single or repeated oral administration (5 days) of 3-BHA, TBQ or 3-TBOQ. The analyses were carried out under conditions which could detect the DNA-adducts produced by reaction of TBQ with calf thymus DNA in vitro. The results suggest that formation of aromatic adducts in vivo by 3-BHA, TBQ or 3-TBOQ in the rat forestomach-DNA is not evident or at least below the detection limits of the current bioassay.

Cancer chemopreventive agents, serratane-type triterpenoids from Picea jezoensis.

Seven serratane-type triterpenoids isolated from the cuticle of Picea jezoensis (Sieb. et Zucc.) Carr. jezoensis (Pinaceae) and the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. hondoensis (Mayer) Rehder (Pinaceae) were studied their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). All compounds showed strong inhibitory effects on the EBV-EA activation, being stronger than that of oleanolic acid, which exerts on cancer preventive activity in animal carcinogenesis models. Among these compounds, 13alpha, 14alpha-epoxy-3beta-methoxyserratan-21beta-ol and 3beta-methoxy-21alpha-hydroxyserrat-14-en-29-al were investigated for the inhibitory effects in a two-stage mouse skin carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. 13alpha,14alpha-Epoxy-3beta-methoxyserratan-21beta-ol was found to exhibit the excellent anti-tumor promoting activity in the in vivo carcinogenesis test.

Craniocervical and aortic atherosclerosis as neurologic risk factors in coronary surgery.

BACKGROUND: Advanced age is associated with increased systemic atherosclerosis and is a consistent neurologic risk factor after coronary artery bypass grafting (CABG). METHODS: We studied prospectively whether varying degrees of a total atherosclerotic score derived from the brain, carotid arteries, and ascending aorta predicted postoperative neuropsychologic (NP) dysfunction and stroke in 177 elderly patients (> or = 60 years) undergoing CABG. RESULTS: Group L (low total atherosclerotic score) had rates of NP dysfunction of 25% and 4%, group I (intermediate) had rates of 33% and 22%, and group H (high) had rates of 79% and 43% on postoperative days 1 and 7, respectively (p < 0.001). The incidence of stroke was higher in group H (14.3%) than in groups I and L (7.8% and 0.9%; p = 0.013). Stepwise logistic regression analysis demonstrated the significant predictors of NP dysfunction on postoperative day 7 to be total atherosclerotic score, peripheral vascular disease, and diabetes mellitus, and those of stroke to be total atherosclerotic score, peripheral vascular disease, and hyperlipidemia. CONCLUSIONS: Perioperative evaluation of craniocervical and aortic atherosclerosis is useful to identify a high-risk patient at postoperative NP dysfunction and stroke after CABG.

Radioiodinated nordiazepam analog for in vivo assessment of benzodiazepine receptors by single photon emission tomography.

2'-Iodo-nordiazepam (2'-IND), a nordiazepam analog iodinated at the 2'-position of the C-5 phenyl ring, was synthesized and evaluated as a potential radiopharmaceutical for investigating brain benzodiazepine receptors by SPECT. [125I]2'-IND was synthesized by the halogen exchange reaction and purified by HPLC. In an in vitro competitive binding study using [3H]diazepam and rat cortical synaptosomol membranes, 2'-IND showed an almost equal affinity for benzodiazepine receptors as diazepam. In a saturation binding study using rat cortical synaptosomal membranes, 2'-IND displayed a Kd of 1.10 nM and a Bmax of 1.87 pmol/mg protein. Biodistribution and metabolism studies in mice showed that [125I]2'-IND exhibited rapid and high accumulation in the brain, and that the cerebral uptake and distribution of this compound occurred in the intact form. Furthermore, the administration of diazepam and flumazenil reduced cortical uptake by approx. 20%, suggesting that the uptake of 2'-IND occurred at least partly in association with benzodiazepine receptors.

Behavior of alpha 2u-globulin accumulating in kidneys of male rats treated with d-limonene: kidney-type alpha 2u-globulin in the urine as a marker of d-limonene nephropathy.

Effects of d-limonene on alpha 2u-globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d-limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type alpha 2u-globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum alpha 2u-globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating alpha 2u-globulin levels were not affected by the d-limonene administration. While the molecular weight of the major alpha 2u-globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type-alpha 2u-globulin was observed in the urine from treated rats. The results were indicative of elimination of alpha 2u-globulin from the kidney to urine, the appearance of kidney-type-alpha 2u-globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the alpha 2u-globulin (kidney-type) in the urine might be used as an indicator of chemically induced alpha 2u-globulin nephropathy.

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA) in the rat (I): Excretion of BHA in urine, feces and expired air and distribution of BHA in the main organs.

The mechanism of the carcinogenic or toxic action of BHA on rat forestomach was examined by studies on the excretion and tissue distribution of radioactivity in F344 male rats given tert-butyl- or methoxy-labelled 3-BHA orally. Within 2 days after a single oral dose of labelled BHA at 1 g/kg body wt, 87-96% of the 14C was excreted, mainly in the urine with smaller amounts in the feces and expired air. More 14C was found in the tissues of rats given the methoxy-labelled compounds. The distributions of 14C in the forestomach and the glandular stomach were similar. At 168 h after treatment, more 14C was found in the forestomach of rats given 2-BHA than in that of rats given 3-BHA. These results indicate that excretion of BHA is rapid, that 4-O-methyl demethylation may take place readily and that demethylated methyl group may become distributed non-specifically in tissues. The carcinogenic or toxic action of BHA on the forestomach does not seem to be due accumulation of BHA in the forestomach.

Differences in alpha 2u-globulins increased in male rat kidneys following treatment with several alpha 2u-globulin accumulating agents: cystein protease(s) play(s) an important role in production of kidney-type-alpha 2u-globulin.

Effects of alpha 2u-globulin accumulating agents on alpha 2u-globulins in rat kidneys were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male animals with decalin (150 mg/kg), 2,2,4-trimethylpentane (50 mg/kg), isophorone (150 mg/kg), d-limonene (150 mg/kg) or 1,4-dichlorobenzene (150 mg/kg) by gavage for 14 consecutive days in each case resulted in a marked intensification of a protein band corresponding to kidney-type-alpha 2u-globulin, with a molecular mass calculated to be approximately 16 kDa. However, intraperitoneal treatment with leupeptin and E-64 (two times 0.07 mmol/kg, for each), well known cystein protease inhibitors, while only slightly increasing this kidney-type-alpha 2u-globulin band, caused the intensification of a approximately 19-kDa molecular mass protein band which was revealed to be a native-type-alpha 2u-globulin by SDS-PAGE and immunoblotting. These results indicated that at least two types of alpha 2u-globulin can be increased in male rat kidney by chemical treatment. Moreover, cystein protease(s) appear(s) to play an important role in the degradation of alpha 2u-globulin and particularly in the conversion of native-type-alpha 2u-globulin to kidney-type-alpha 2u-globulin in rat kidneys.

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole (2- and 3-BHA) in the rat. (II): Metabolism in forestomach and covalent binding to tissue macromolecules.

The mechanism of action of 2(3)-tert-butyl-4-hydroxyanisole (2-BHA or 3-BHA) on rat forestomach epithelium was studied by examining the metabolites of BHA in the stomach and the covalent binding of BHA to macromolecules in the forestomach epithelium. Male F344 rats 6 weeks old were given a single intragastric injection of 1 g/kg body wt of [tert-14C]-3-BHA (Bu-3-BHA) or [methyl-14C]-3-BHA (Me-3-BHA), and 6 h later BHA metabolites in the forestomach, glandular stomach and stomach contents were examined by thin-layer chromatography. No significant amounts of metabolites were detected in the forestomach or glandular stomach epithelium and almost all the radioactivity in these tissues was extracted with organic solvents. In in vitro experiments also, no significant amounts of metabolites were detected when the 9000 g supernatant of the forestomach or glandular stomach epithelium, or gastric juice was incubated with Bu-3-BHA in the absence or presence of NADPH. In binding studies, rats were given Bu-3-BHA, [tert-14C]-2-BHA (Bu-2-BHA), Me-3-BHA or [methyl-14C] butylated hydroxytoluene (Me-BHT) intragastrically at a dose of 1 g/kg body wt with or without pretreatment with unlabelled 1% 3-BHA or BHT in the diet for 6 days. Six hours after treatment with a labelled compound, the rats were sacrificed and the DNA, RNA and protein of their forestomach, glandular stomach, liver and kidney were isolated. Bu-3-BHA, Bu-2-BHA and Me-3-BHA did not bind covalently to forestomach DNA or RNA, and the amounts of radioactivity of these compounds bound to proteins in the 4 tissues were similar. These findings suggest that BHA acts on the forestomach epithelium directly without metabolic activation, and that its action is not related to its binding to DNA or RNA.

Metabolism of 2- and 3-tert-butyl-4-hydroxyanisole in the rat (III): Metabolites in the urine and feces.

The urinary and fecal metabolites of orally administered 2-tert-butyl-4-hydroxyanisole (2-BHA) and 3-tert-butyl-4-hydroxyanisole (3-BHA) in rats were identified. Samples of 2-day pooled urine and feces of rats given a single intragastric dose of 1 g/kg body wt of tert[butyl-14C]3-BHA (*Bu-3-BHA). tert[butyl-14C]2-BHA (*Bu-2-BHA), [methyl-14C]3-BHA (*Me-3-BHA) or [methyl-14C]-2-BHA (*Me-2-BHA) were analyzed by comparing thin-layer chromatography (TLC) retentions with authentic standards. Conjugated metabolites were identified after enzymatic hydrolysis. Proton magnetic resonance spectroscopy and electron impact mass spectrometry were used for confirmation of the authentic standards. In rats given 3-BHA, a major metabolite in the urine was 3-BHA-glucuronide with a smaller amount of tert-butylhydroquinone (TBHQ)-sulfate, while unchanged 3-BHA and 3-BHA-glucuronide were detected in the feces. In rats given 2-BHA, the main metabolites were the sulfate conjugates of 2-BHA, 4-tert-butyl-5-methoxy-1,2-benzoquinone (2-TBOQ) and the glucuronide of 2-BHA in the urine, while unchanged 2-BHA was found in the feces.

Radioiodinated 2'-iodospiperone: a new radioligand for in vivo dopamine receptor study.

In vivo dopamine receptor binding of the newly synthesized ligand, 125I-2'-iodospiperone (125I-2'-ISP), was studied in mouse brain. The highest accumulation was found in the striatum. Analysis of the striatal homogenate showed the 125I-2'-ISP to be metabolically stable. Furthermore, this striatal binding was saturable and displaced only by dopaminergic drugs. On the other hand, the accumulation in the cortex was as low as that of the cerebellum and uneffected by the administration of serotoninergic drugs and dopaminergic drugs; results assessed by macroautoradiographic studies. Thus, the newly synthesized 125I-2'-ISP presented high affinity for dopamine receptors in vivo and therefore, holds great potential for the in vivo dopamine receptor studies, provided 123I becomes readily available.

In vitro evaluation of radioiodinated butyrophenones as radiotracer for dopamine receptor study.

Radioiodinated butyrophenone compounds are attracting the interest of those working on dopamine receptor studies; structure-activity relationship study has revealed the ortho position of the p-fluorobutyrophenone moiety as a very plausible iodination site. Various synthesized butyrophenones iodinated at the ortho position of p-fluorobutyrophenone moiety, 2'-iodohaloperidol (2'-IHP), 2'-iodotrifluperidol (2'-ITP) and 2'-iodospiperone (2'-ISP) were tested for their abilities to inhibit 3H-spiperone (SP) binding for the dopamine (D-2) receptor, together with reference compounds (SP, haloperidol(HP) and 4-iodospiperone (4-ISP]. The order of binding affinity of the tested compounds was SP greater than 2'-ISP greater than HP greater than 4-ISP greater than 2'-IHP greater than 2'-ITP. Whereas, the serotonin (S-2) receptor binding affinity of SP and its iodinated analogues were in the order of SP much greater than 4-ISP greater than 2'-ISP. Furthermore, in the saturation binding study using the striatal membrane preparations, the 2'-ISP displayed a KD of 0.25 nM with maximum number of binding site Bmax of 210 fmol/mg protein. These data indicated the 2'-ISP as holding high affinity for dopamine receptors and a low affinity for serotonin receptors. Thus, the 125I-2'-ISP was a very potent radioligand for in vitro dopamine (D-2) receptor studies, and 123I-2'-ISP holds very promising characteristics as for in vivo dopamine receptor studies, as well.

Benzodiazepines and their metabolites: relationship between binding affinity to the benzodiazepine receptor and pharmacological activity.

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.

Long-term mild hypothermia with extracorporeal lung and heart assist improves survival from prolonged cardiac arrest in dogs.

BACKGROUND AND PURPOSE: although normothermic extracorporeal lung and heart assist (ECLHA) improves cardiac outcomes, patients can not benefit from hypothermia-mediated brain protection. The present study evaluated the effects of long-term ECLHA with mild to moderate hypothermia (33 degrees C) in a canine model of prolonged cardiac arrest. METHODS: 15 dogs were assigned to either the hypothermic (seven dogs, 33 degrees C) or normothermic group (eight dogs, 37.5 degrees C). All dogs were induced to normothermic ventricular fibrillation (VF) for 15 min, followed by 24 h of ECLHA and 72 h of intensive care. The hypothermia group maintained core (pulmonary artery) temperature at 33 degrees C for 20 h starting from resuscitation, then were rewarmed by 28 h. Outcome evaluations included: (1) mortality; (2) catecholamine dose; (3) time to extubation; (4) necrotic myocardial mass (g); and (5) neurological deficits score (NDS). RESULTS: in the normothermic group five dogs died of cardiogenic shock and one dog succumbed to poor oxygenation. The two surviving dogs remained comatose (NDS 60.5 +/- 4.9%) with necrotic myocardial mass of 14.5 +/- 3.5 g. In the hypothermic group, one dog died from pulmonary dysfunction, the other six dogs survived. The surviving dogs showed brain damage (29.8 +/- 2.5%), but there was evidence of some brain-protective effect. The mass of necrotic myocardium was 4.2 +/- 1.3 g in the hypothermic group or 3.4 times smaller than in the normothermic group. The survival rate was significantly higher in the hypothermic than in the normothermic group (P < 0.05). The catecholamine requirement was also lower in the hypothermic than in the normothermic dogs (P < 0.05). CONCLUSIONS: Long-term mild to moderate hypothermia with ECLHA induced immediately after cardiac arrest improved survival as well as cerebral and cardiac outcomes.

Distribution and metabolism of F6-1,25(OH)2 vitamin D3 and 1,25(OH)2 vitamin D3 in the bones of rats dosed with tritium-labeled compounds.

26,26,26,27,27,27-Hexafluo-1,25(OH)2 vitamin D3, the hexafluorinated analog of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biologic activity in the kidneys and small intestine appears to be related to F6-1,25(OH)2 vitamin D3 metabolism to ST-232, 26,26,26,27,27,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3, a bioactive 23S-hydroxylated form that is resistant to further metabolism. Since F6-1,25(OH)2 vitamin D3 is considered to prevent osteoporotic decrease in bone mass by suppressing bone turnover, we here compared the distribution and metabolism of [1 beta-3H]F6-1,25(OH)2 vitamin D3 and [1 beta-3H]1,25(OH)2 vitamin D3 in bones of rats by autoradiography and radio-HPLC. In the dosed groups, radioactivity was detected locally in the metaphysis, the modeling site in bones. As compared with the [1 beta-3H]1,25(OH)2 vitamin D3 case, [1 beta-3H]F6-1,25(OH)2 vitamin D3 was significantly retained in this site, and moreover, it mainly persisted as unchanged compound and ST-232. These findings indicate that the reason for the higher potency of F6-1,25(OH)2 vitamin D3 than 1,25(OH)2 vitamin D3 in bones are linked with increased distribution and reduced metabolism.

Ethylnitrosourea-induced mutation and molecular analysis of transgenic mice containing the gpt shuttle vector.

Novel transgenic mice were developed in order to study the in vivo mutagenesis. The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda. The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E. coli by an in vitro packaging method and is selectable as a Kanr phenotype. Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG). In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene. Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro. A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines. To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses. The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested. ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical. The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively. The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction. The amplified DNA was subject to DNA sequence analysis. Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants. DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants. These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene.

Development of a novel CHL/IU cell line with an incorporated gpt shuttle vector for concurrent analysis of gene mutations and chromosome aberrations.

A cosmid shuttle vector containing the target gene of Escherichia coli gpt coding xanthine-guanine phosphoribosyl transferase was constructed. The shuttle vector was designed to be rescued into the gpt-deficient Escherichia coli from Chinese hamster CHL/IU cells through an in vitro packaging method. Mutations occurred at the target gene can be detected with a selective agent, 6-thioguanine (6-TG). The shuttle vector was stably transfected into CHL/IU cells to give several cell lines containing copies of the shuttle vector in the chromosomes. Each cell line exhibited a characteristic rescue efficiency (0 to 1.9 x 10(5) CFU/microgram of genomic DNA) of the shuttle vector and spontaneous mutation frequency (3.9 x 10(-5) to over 10(-2)) at the 6-TG selection. One transgenic cell line (KN63), which showed a higher rescue efficiency and a low spontaneous mutation frequency, was selected and tested for the ability to respond to a genotoxic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG increased both the mutation frequency at the target gene and the number of the cells with chromosome aberrations. DNA sequence analysis of 6-TG mutants showed that predominant mutations (10/14) were identified as G:C to A:T transitions in MNNG-induced mutants, whereas transversions were predominant (5/9) in spontaneous mutants. These results suggest that this transgenic CHL/IU cell line can be a useful tool for analyzing the relation between gene mutations and chromosome aberrations.