Delta Mice Research Articles

Assessment of the mutagenic potential of arecoline in gpt delta transgenic mice

Chewing the areca nut is carcinogenic to humans. Arecoline, a major alkaloid in areca nut, is suspected to be a carcinogenic component. It has been shown to have genotoxic potential in various in vitro systems; but information on its in vivo genotoxicity is limited. To investigate the organ-specific mutagenic potential of arecoline, we employed gpt delta transgenic mice to analyze the mutagenicity of arecoline in the oral tissues and liver. Male gpt delta mice were given arecoline hydrobromide in drinking water at 300 and 700μg/mL for 6 weeks. 4-Nitroquinoline-1 (4-NQO) was used as a positive control. Two weeks after the last treatment, mutation frequencies in the oral tissues (a mixture of gingival, buccal, pharyngeal and sublingual tissue) and liver were detected and mutation spectra were analyzed. There were no statistically significant differences in the average mutation frequencies between arecoline-treated and untreated groups in both the oral tissues and liver. However, in the oral tissues, one mouse in arecoline-300μg/mL group and two mice in arecoline-700μg/mL group showed more than 2.5-fold higher mutation frequencies than the untreated group; they also exhibited unique mutation spectra compared to spontaneous mutation types. In these three mice, all mutations occurred at G:C sites, where G:C→T:A transversions were most frequent, followed by G:C→A:T transitions and G:C→C:G transversions. The main type of spontaneous mutation in both the oral tissues and liver was G:C→A:T transition. These results suggest that arecoline poses a mutagenic hazard in the oral tissues of gpt delta transgenic mice.

Dammar resin, a non-mutagen, inducts oxidative stress and metabolic enzymes in the liver of gpt delta transgenic mouse which is different from a mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline

Dammar resin has long been used in foods as either a clouding or a glazing agent. In a recent study, 2% Dammar resin showed significant hepatocarcinogenicity in a rat 2-year bioassay. Therefore, for an accurate estimate of human risk, it is necessary to understand whether Dammar resin induces liver genotoxicity and the underlying mechanisms of its hepatocarcinogenicity. Modifying effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a typical genotoxic carcinogen produced during cooking of protein-rich foods, was also studied in the present study. Exposure of gpt delta mice to Dammar resin at a dose of 2% for 12 weeks did not induce any obvious mutagenicity in the liver. However, the index of cell proliferation, the level of 8-OHdG, and bax, bcl-2, p53, cyp1a2, cyp2e1, gpx1 and gstm2 gene expression were all significantly increased when compared with the control group. In the IQ treatment group, at a dose of 300ppm, mutagenicity was readily detected, the index of cell proliferation increased, and p53, cyp2e1 and gpx1 gene expression was down-regulated in the liver. Down-regulation of p53, P450s, and gpx1 in the livers of IQ treated mice are consistent with its genotoxic mechanism of carcinogenicity observed in a 675-day study. In contrast, our results using gpt delta mice suggest that Dammar resin is not genotoxic. Instead, the Dammar resin-induced hepatocarcinogenicity seen in our previous 2-year study with rats may have been mediated by non-genotoxic mechanisms, including increased P450 enzyme activity, increased oxidative stress, altered gene expression, and promotion of cell proliferation.

The application of hepatic P450 reductase null gpt delta mice in studying the role of hepatic P450 in genotoxic carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mutagenesis

The cytochrome P450 (P450 or CYP) is involved in both detoxification and metabolic activation of many carcinogens. In order to identify the role of hepatic P450 in the mutagenesis of genotoxic carcinogens, we generated a novel hepatic P450 reductase null (HRN) gpt delta mouse model, which lacks functional hepatic P450 on a gpt delta mouse background. In this study, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was used to treat HRN gpt delta mice and control littermates. Gene mutations in the liver and lungs were detected, and mutation spectra were analyzed. Pharmacokinetic analyses were performed, and tissue levels of NNK and metabolite were determined. NNK-induced mutant frequencies (MFs) were equivalent to spontaneous MFs in the liver, but increased more than 3 times in the lungs of HRN gpt delta mice compared to control mice. NNK-induced mutation spectra showed no difference between HRN gpt delta mice and control littermates. Toxicokinetic studies revealed reduced clearance of NNK with elevated tissue concentrations in HRN gpt delta mice. To our knowledge, these are the first data demonstrating that NNK cannot induce mutagenesis in the liver without P450 metabolic activation, but can induce mutagenesis in lungs by a hepatic P450-independent mechanism. Moreover, our data show that hepatic P450 plays a major role in the systemic clearance of NNK, thereby protecting the lungs against NNK-induced mutagenesis. Our model will be useful in establishing the role of hepatic versus extrahepatic P450-mediated mutagenesis, and the relative contributions of P450 compared to other biotransformation enzymes in the genotoxic carcinogens' activation.

Reporter gene mutation in the livers of <i>gpt</i> delta mice treated with 5-(hydroxymethyl)-2-furfural, a contaminant of various foods

A major product formed during the Maillard reaction is 5-(hydroxymethyl)-2-furfural (HMF), which is present in various foods and beverages such as honey and fruit juice. HMF was shown to be a hepatocarcinogen in female mice using long-term bioassays. Although HMF is not a mutagen in conventional in vitro mutation assays, 5-sulfoxymethylfurfural (SMF), a reactive metabolite of HMF produced following sulfotransferase conjugation, does show mutagenicity. Thus, HMF-induced hepatocarcinogenesis likely involves genotoxic mechanisms. To clarify the mechanisms underlying HMF-induced hepatocarcinogenesis, female B6C3F(1) gpt delta mice were given HMF at carcinogenic doses (188 or 375 mg/kg b.w.) by gavage for 5 days per week for 4 weeks. This treatment produced no significant differences in mutant frequencies (MFs) of gpt and red/gam (Spi(-)) genes among the groups. These results suggest that genotoxicity does not contribute to HMF-induced hepatocarcinogenesis. Parameters related to cell proliferation, such as proliferation cell nuclear antigen-labeling index and Cyclin D1 and E1 mRNA expression, exhibited no significant changes in the livers of HMF-treated groups. In view of the lack of carcinogenicity in rats, HMF may be considered to be a weak carcinogen. These results help us to understand the underlying mechanisms of action of HMF carcinogenesis.

<i>In vivo</i> genotoxicity of 1-methylnaphthalene from comprehensive toxicity studies with B6C3F1 <i>gpt</i> delta mice

1-Methylnaphthalene (1-MN), a constituent of the polycyclic aromatic hydrocarbons (PAHs), is a lung carcinogen in mice. However, conventional genotoxicity tests such as the Ames test and sister chromatid exchange (SCE) test have yielded equivocal results. In the present study, the in vivo genotoxicity of 1-methylnaphthalene (1-MN) together with its toxicological profile was investigated in a 13-week repeated dose toxicity study of 1-MN using B6C3F1 gpt delta mice. In the serum biochemistry, significant increases in AST and ALP were observed in males of the 0.15% 1-MN group. From histopathological examination, the incidence of single cell necrosis in the liver was significantly increased in males of the 0.15% 1-MN group; however, no changes were observed in the lungs, the target organ of 1-MN. In an in vivo mutation assay, no changes in mutant frequencies of gpt and red/gam (Spi-) in lung DNA of 1-MN treated mice were observed at 13 weeks. In addition, there were no significant differences in the proliferating cell nuclear antigen (PCNA)-positive ratios in bronchiolar epithelial cells among the groups for either sex. These results suggest that 1-MN at a carcinogenic dose not induce overt toxicity for any organs and has no in vivo genotoxicity in the lungs.

Lack of Mutagenicity of SMD-502, a New Vitamin D3 Analog for Topical Application, in Skin and Liver of gpt delta Transgenic Mice and in GDL1 Cells

SMD-502, a new vitamin D3 (VD3) analog, is an antipsoriatic drug candidate. The compound exhibited fewer side effects than known VD3 analogs in animal models, probably because the compound is rapidly converted into pharmacologically inactive form after permeating into systemic circulation from the application site on skin. This feature of the compound makes it difficult to assess genotoxic risk in vivo with the standard approach of bone marrow or peripheral blood micronucleus assays in rodents because of the low blood concentration level. To evaluate in vivo mutagenicity of the compound in the present study, mutant frequency (MF) in skin and liver of gpt delta transgenic mice was examined with percutaneous administration of SMD-502 for 28 days. In tissues collected 7 days after the end of administration, no significant increase in the MF was observed in either skin or liver. Additionally, when the compound was tested in a GDL1 cell line established from gpt delta mice, GDL1 cells exhibited no significant increase in MFs even under conditions in which they would be exposed to a much higher concentration of the compound than in the in vivo study. The results in this study further supported the consideration that SMD-502 has no mutagenic activity.

Abstract P183: Cardiac Deletion of CaMKII Delta and Gamma Protects Against Heart Failure Despite Activation of Calcineurin Signaling

CaMKII delta and gamma are the major CaMKII genes expressed in the heart, and both are up-regulated in response to pressure overload. Recently, we have demonstrated that CaMKII delta single knockout mice are protected against cardiac hypertrophy and remodeling. However, the role of CaMKII gamma and potential redundant functions of CaMKII delta and gamma are still elusive. The aim of the present study was to evaluate the function of CaMKII delta and gamma by a cardiomyocyte-specific double knockout mouse model(delta/gamma-CKO). Strikingly, whereas delta and gamma single knockout mice displayed only slightly reduced levels of cardiac phospholamban (PLN) phosphorylation at the CaMKII phosphorylation site threonin 17, in delta/gamma-CKO mice there was almost no residual PLN-threonin-17 phosphorylation detectable. Surprisingly and in contrast to delta and gamma single knockout mice, delta/gamma-CKO mice did develop cardiac hypertrophy after transverse aortic constriction (TAC). Despite cardiac hypertrophy we observed markedly reduced cardiac fibrosis and apoptosis. Microarray analysis revealed a distinct different gene expression profile pointing to an activation of calcineurin in delta/gamma-CKO mice after TAC. Phosphorylation of calcineurin at serine 197, which leads to an inactivation of its enzymatic activity, was almost abolished in delta/gamma-CKO mice. To test the therapeutical implications of a complete myocardial CaMKII knockout, an tamoxifen-inducible knockout system was established. Knockout of CaMKII delta and gamma was induced by administration of tamoxifen three weeks after TAC surgery. Whereas control mice did develop overt heart failure and cardiac remodeling 16 weeks after TAC, delta/gamma-iCKO mice recovered from cardiac dysfunction. Taken together, our mouse genetic studies demonstrate that CaMKII delta and gamma are promising drug targets to restore cardiac function after pathological stress. These data also unmask a cross talk of CaMKII to endogenous calcineurin signaling, which results in adaptive cardiac hypertrophy and not pathological remodeling.

In vivo mutagenicity and DNA damage in the lungs, livers, and kidneys of gpt delta mice treated with acrylamide

In vivo mutagenicity and DNA damage in the lungs, livers, and kidneys of gpt delta mice treated with acrylamide

Antigenotoxic effects of p53 on spontaneous and ultraviolet light B–induced deletions in the epidermis of gpt delta transgenic mice

Tumor development in the skin may be a multistep process where multiple genetic alterations occur successively. The p53 gene is involved in genome stability and thus is referred to as "the guardian of the genome." To better understand the antigenotoxic effects of p53 in ultraviolet light B (UVB)-induced mutagenesis, mutations were measured in the epidermis of UVB-irradiated p53(+/+) and p53(-/-) gpt delta mice. In the mouse model, point mutations and deletions are separately identified by the gpt and Spi(-) assays, respectively. The mice were exposed to UVB at single doses of 0.5, 1.0, or 2.0 kJ/m(2) . The mutant frequencies (MFs) were determined 4 weeks after the irradiation. All doses of UVB irradiation enhanced gpt MFs by about 10 times than that of unirradiated mice. There were no significant differences in gpt MFs and the mutation spectra between p53(+/+) and p53(-/-) mice. The predominant mutations induced by UVB irradiation were G:C to A:T transitions at dipyrimidines. In contrast, in unirradiated p53(-/-) mice, the frequencies of Spi(-) large deletions of more than 1 kb and complex-type deletions with rearrangements were significantly higher than those of the Spi(-) large deletions in p53(+/+) counterparts. The specific Spi(-) mutation frequency of more than 1 kb deletions and complex types increased in a dose-dependent manner in the p53(+/+) mice. However, no increase of such large deletions was observed in irradiated p53(-/-) mice. These results suggest that the antigenotoxic effects of p53 may be specific to deletions and complex-type mutations induced by double-strand breaks in DNA.

Radiation Dose-Rate Effect on Mutation Induction in Spleen and Liver of gpt delta Mice

The effect of dose rate on radiation-induced mutations in two somatic tissues, the spleen and liver, was examined in transgenic gpt delta mice. These mice can be used for the detection of deletion-type mutations, and these are the major type of mutation induced by radiation. The dose rates examined were 920 mGy/min, 1 mGy/min and 12.5 microGy/min. In both tissues, the number of mutations increased with increasing dose at each of the three dose rates examined. The mutation induction rate was dependent on the dose rate. The mutation induction rate was higher in the spleen than in the liver at the medium dose rate but was similar in the two tissues at the high and low dose rates. The mutation induction rate in the liver did not show much change between the medium and low dose rates. Analysis of the molecular nature of the mutations indicated that 2- to 1,000-bp deletion mutations were specifically induced by radiation in both tissues after high- and low-dose-rate irradiation. The occurrence of deletion mutation without any sequence homology at the break point was elevated in spleen after high-dose-rate irradiation. The results indicate that the mutagenic effects of radiation in somatic tissues are dependent on dose rate and that there is some variability between tissues.

Enhancing effects of carbon tetrachloride on in vivo mutagenicity in the liver of mice fed 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)

Chronic stimulus subsequent to cell injury plays an important role in cancer development, but the precise mechanisms remain unknown partly because appropriate animal models are lacking. In the present study, the effects of hepatotoxicant carbon tetrachloride (CCl(4)) on in vivo mutagenicity were investigated using gpt delta mice with or without p53. Female B6C3F(1) p53-proficient or -deficient gpt delta mice were given a diet containing 300 ppm of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) for 13 weeks, concurrently with intraperitoneal injection of 1 ml/kg CCl(4) solution once a week. Mutant frequencies of gpt and red/gam in p53-proficient mice fed MeIQx were both significantly elevated by CCl(4)co-treatment. Enhancing effects of CCl(4) treatment were also noted in p53-deficient mice. In the mutation spectra analysis of gpt mutant colonies, G:C to T:A transversions were predominantly observed regardless of CCl(4) injection, and clonal expansion of gpt colonies were increased in the co-treated group as compared with MeIQx alone group. The present data showing no significant changes in mRNA expression levels of CYP1A2 and GSTa4 between MeIQx-treated groups with and without CCl(4). In the Western blotting analysis, CYP1A2 protein levels were significantly decreased in the co-treated group as compared to MeIQx alone group, and GSTα protein levels were not changed among any groups. It is suggested that the mutant frequency by co-treatment with CCl(4) might result from some factors other than p53 or MeIQx metabolism/excretion. Thus, our data clearly demonstrate that this model could be a powerful tool for identifying the mechanisms underlying combinatorial effects on carcinogenesis.

Effects of co-treatment of dextran sulfate sodium and MeIQx on genotoxicity and possible carcinogenicity in the colon of p53-deficient mice

To investigate the effects of dextran sulfate sodium (DSS) and/or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) on in vivo genotoxicity in the colon, male C57BL/6 p53 (+/+), p53 (+/-) or p53 (-/-) gpt delta mice were twice given 1-week treatment with DSS, 2 weeks apart, and then sacrificed after 2 and 14 weeks. Although colon length was significantly shortened after DSS treatment in all genotypes at each time point, no significant difference in gpt mutant frequency (MF) and tumorigenicity was found between DSS and control groups regardless of genotype. Then, male B6C3F(1) p53 (+/+) or p53 (+/-) gpt delta mice were given DSS as described above and/or fed 300 ppm MeIQx for 7 weeks. Colon length was significantly shortened with DSS in either genotype at weeks 7 and 26, but no effects of co-treatment with MeIQx or p53 deficiency were evident. MeIQx showed a tendency to increase gpt MF in the colon of mice with either genotype, but co-treatment with DSS did not affect these increments. Appreciable incidences of colonic aberrant crypt foci (ACFs) were reported in DSS as well as co-treatment groups of each genotype. Colonic adenomas were observed in co-treatment groups of both genotypes as well as the DSS-only group of p53 (+/+). No effects of the combination of DSS and MeIQx on colon pre- and neoplastic lesions were reported. Our results indicate that MeIQx may take more than 7 weeks to induce genotoxicity in the colon and that the co-treatment of mice did not enhance colon tumorigenicity even in p53-deficient mice.

δ Receptors Are Required for Full Inhibitory Coupling of μ Receptors to Voltage-Dependent Ca2+ Channels in Dorsal Root Ganglion Neurons

Recombinant micro and delta opioid receptors expressed in cell lines can form heterodimers with distinctive properties and trafficking. However, a role for opioid receptor heterodimerization in neurons has yet to be identified. The inhibitory coupling of opioid receptors to voltage-dependent Ca(2+) channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. We examined micro-receptor coupling to VDCCs in dorsal root ganglion neurons of delta(+/+), delta(+/-), and delta(-/-) mice. Neurons deficient in delta receptors exhibited reduced inhibition of VDCCs by morphine and [D-Ala(2),Phe(4),Gly(5)-ol]-enkephalin (DAMGO). An absence of delta receptors caused reduced efficacy of DAMGO without affecting potency. An absence of delta receptors reduced neither the density of VDCCs nor their inhibition by either the GABA(B) receptor agonist baclofen or intracellular guanosine 5'-O-(3-thio)triphosphate. Flow cytometry revealed a reduction in micro-receptor surface expression in delta(-/-) neurons without altered DAMGO-induced internalization. There was no change in micro-receptor mRNA levels. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)-sensitive mu-receptor-coupling efficacy was fully restored to delta(+/+) levels in delta(-/-) neurons by expression of recombinant delta receptors. However, the dimerization-deficient delta-15 construct expressed in delta(-/-) neurons failed to fully restore the inhibitory coupling of micro-receptors compared with that seen in delta(+/+) neurons, suggesting that, although not essential for micro-receptor function, micro-delta receptor dimerization contributes to full micro-agonist efficacy. Because DAMGO exhibited a similar potency in delta(+/+) and delta(-/-) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis.

Inhibition of thalamic excitability by 4,5,6,7‐tetrahydroisoxazolo[4,5‐c]pyridine‐3‐ol: a selective role for δ‐GABAA receptors

The sedative and hypnotic agent 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP) is a GABA(A) receptor (GABA(A)R) agonist that preferentially activates delta-subunit-containing GABA(A)Rs (delta-GABA(A)Rs). To clarify the role of delta-GABA(A)Rs in mediating the sedative actions of THIP, we utilized mice lacking the alpha(1)- or delta-subunit in a combined electrophysiological and behavioural analysis. Whole-cell patch-clamp recordings were obtained from ventrobasal thalamic nucleus (VB) neurones at a holding potential of -60 mV. Application of bicuculline to wild-type (WT) VB neurones revealed a GABA(A)R-mediated tonic current of 92 +/- 19 pA, which was greatly reduced (13 +/- 5 pA) for VB neurones of delta(0/0) mice. Deletion of the delta- but not the alpha(1)-subunit dramatically reduced the THIP (1 mum)-induced inward current in these neurones (WT, -309 +/- 23 pA; delta(0/0), -18 +/- 3 pA; alpha(1) (0/0), -377 +/- 45 pA). Furthermore, THIP selectively decreased the excitability of WT and alpha(1) (0/0) but not delta(0/0) VB neurones. THIP did not affect the properties of miniature inhibitory post-synaptic currents in any of the genotypes. No differences in rotarod performance and locomotor activity were observed across the three genotypes. In WT mice, performance of these behaviours was impaired by THIP in a dose-dependent manner. The effect of THIP on rotarod performance was blunted for delta(0/0) but not alpha(1) (0/0) mice. We previously reported that deletion of the alpha(1)-subunit abolished synaptic GABA(A) responses of VB neurones. Therefore, collectively, these findings suggest that extrasynaptic delta-GABA(A)Rs vs. synaptic alpha(1)-subunit-containing GABA(A)Rs of thalamocortical neurones represent an important molecular target underpinning the sedative actions of THIP.

Spontaneous and Induced gpt and Spi− Mutant Frequencies in gpt delta Transgenic Rodents

Transgenic rodent mutation assays are useful models for investigating the genotoxicity of chemicals in vivo. Transgenic gpt delta mice contain multiple copies of chromosomally integrated lambda EG10 phage shuttle vector, which contains reporter genes that allow detection of mutations. This system can identify both point mutations by the gpt assay (6-thioguanine selection) and certain types of deletions using the Spi− assay. Transgenic gpt delta rats, which have the same lambda EG10 DNA copies as gpt delta mice, have also been developed. The average spontaneous gpt mutant frequency (MF) in both gpt delta mice and rats is approximately 4.5×10-6. In the Spi− assay, the average spontaneous Spi− MF is approximately 2.7×10-6 in gpt delta mice, similar to that of gpt delta rats. More than 20 chemicals and irradiations have been analyzed with these systems, and this review summarizes the MFs and treatment conditions. The data demonstrate that these transgenic rodent models are useful for detection and analysis of point mutations and deletions in vivo.

Spontaneous Mutagenesis in Rodents: Spontaneous Gene Mutations Identified by Neutral Reporter Genes in gpt Delta Transgenic Mice and Rats

Transgenic rodents are valuable models for investigation of genotoxicity of chemicals in vivo. We have developed gpt delta transgenic mice (C57BL/6J background) and rats (Sprague-Dawley, SD), which have the ability to identify both point mutations by the gpt assay [6-thioguanine (6-TG) selection] and certain types of deletions by the Spi- (Spi, sensitive to P2 interference) assay. Recently, the gpt delta SD rat was backcrossed with the Fisher 344 (F344) rat to establish an gpt delta F344 rat. The average spontaneous gpt mutation frequencies (MFs) are about 4.5×106 in both SD and F344 gpt delta rats as well as in gpt delta mice. The G:C to A:T transitions at 5'-CpG-3'sites and G:C to T:A transversions are the predominant spontaneous gpt mutations in rats and mice. However, there is one false mutation (e.g. A:T to T:A at position 299) in the rats. The base substitution may have arisen when the lambda EG10 transgene was introduced into the genome of the SD rat during transgenesis. In the Spi- assay, 1-bp deletions in repetitive sequences are predominantly observed in both mice and rats. Possible mechanisms underlying the spontaneous mutations in gpt delta rodents are discussed.

The expression of GABAAβ subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells

The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABA(A) receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.

Tolerance induction to self-MHC antigens in fetal and neonatal mouse B cells

We have studied the mechanisms of tolerance induction to self-MHC antigens in mouse B cells during fetal development and the post-natal period. To monitor the fate of autoreactive B cell clones, we used the 3-83 micro delta B cell receptor (BCR)-transgenic (Tg) and -knock-in (KI) mouse models. These BCR-Tg and -KI B cells recognize the MHC class I molecules H-2K(k) and H-2K(b), with a high or moderate affinity, respectively. We compared the fate of BCR-Tg and -KI B cells in H-2K(b)-bearing animals and H-2K(b)-negative controls at various stages of their fetal development and post-natal life. Our data show that, in contrast to what occurs in adult B cells, anergy is the main component of tolerance induction in 3-83 micro delta BCR-Tg K(b+) autoreactive fetuses, while 3-83 BCR-KI fetuses primarily use receptor editing. Interestingly, autoreactive B cell deletion is absent or merely marginal before birth. Our results indicate that tolerance induction is effective as early as embryonic day 16.5 and that in the fetus and neonate, like in the adult, the main mechanism of B cell tolerance functioning in the 3-83 KI system is receptor editing. In contrast, in the 3-83 micro delta mice where receptor editing is hindered, adult and fetal B cells differ in their preferential use of mechanisms leading to self-tolerance (i.e. deletion versus anergy).

Mutations in the lungs of gpt delta transgenic mice following inhalation of diesel exhaust

Diesel exhaust (DE) is a major airborne pollutant of urban areas. It contains various polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs. In this study, gpt delta mice were treated with inhalation of 1 or 3 mg m(-3) DE, or a single intratracheal instillation of diesel exhaust particles (DEP) or DEP extract. In the lungs of mice treated with inhalation of 3 mg m(-3) DE for 12 weeks, the mutant frequency (MF) was 3.2-fold higher than that of the control group (1.90 x 10(-5) and 0.59 x 10(-5), respectively). An instillation of DEP and DEP extract resulted in a significant dose-dependent linear increase in MF. In mice treated with 0.5 mg DEP and 0.2 mg DEP extract, the MFs were 3.0- and 2.7-fold higher than that of the control group, respectively. The mutagenic potency (MF mg(-1)) of DEP extract (5.6 x 10(-5)) was double that of DEP (2.7 x 10(-5)), suggesting that the mutagenicity of the latter is derived primarily from compounds in the extract, which itself is responsible for ca. 50% of the weight of DEP. G:C-->A:T transitions were the predominant gpt mutation induced by all three treatments and G:C-->T:A transversions were induced by DEP and DEP extract. Guanine bases centered in nucleotide sequences such as GGA, TGA, CGG, and CGT were the major mutation targets of all three treatments. Thus, our results suggest that the mutagens contained in DEP such as PAH and nitrated PAHs induce mutations and may be responsible for carcinogenesis caused by inhalation of DE.

Detection of oxidative DNA damage, cell proliferation and in vivo mutagenicity induced by dicyclanil, a non-genotoxic carcinogen, using gpt delta mice

To ascertain whether measurement of possible contributing factors to carcinogenesis concurrently with the transgenic mutation assay is useful to understand the mode of action underlying tumorigenesis of non-genotoxic carcinogens, male and female gpt delta mice were given dicyclanil (DC), a mouse hepatocarcinogen showing all negative results in various genotoxicity tests, at a carcinogenic dose for 13 weeks. Together with gpt and Spi − mutations, thiobarbituric acid-reactive substances (TBARS), 8-hydroxydeoxyguanosine (8-OHdG) and bromodeoxyuridine labeling indices (BrdU-LIs) in the livers were examined. Whereas there were no changes in TBARS levels among the groups, significant increases in 8-OHdG levels and centrilobular hepatocyte hypertrophy were observed in the treated mice of both genders. In contrast, BrdU-LIs and liver weights for the treated females, but not the males were significantly higher than those for the controls. Likewise, the gpt mutant frequencies (MFs) in the treated females were significantly elevated, GC:TA transversion mutations being predominant. No significant alterations were found in the gpt MFs of the males and the Spi − MFs of both sexes. The results for the transgenic mutation assays were consistent with DC carcinogenicity in terms of the sex specificity for females. Considering that 8-OHdG induces GC:TA transversion mutations by mispairing with A bases, it is likely that cells with high proliferation rates and a large amounts of 8-OHdG come to harbor mutations at high incidence. This is the first report demonstrating DC-induced genotoxicity, the results implying that examination of carcinogenic parameters concomitantly with reporter gene mutation assays is able to provide crucial information to comprehend the underlying mechanisms of so-called non-genotoxic carcinogenicity.