A search for radiosensitive mouse mutants by use of the micronucleus technique

In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel (Slcon), scabby (scb), viable dominant spotting (Wv), quaking (qk), fidget (fi) and postaxial hemimelia (px), heterozygous lurcher (Lc), hemizygous gyro (Gy), the compounds Slcon/grizzle-belly (SlgbH) and Wv/rump-white (Rw) and MS/Ae mice, were regarded as radiosensitive, with Slcon/Slcon the highest in rank order. Homozygous wabbler-lethal (wl) and wasted (wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.     

Micronucleus induction in mouse bone marrow by phenacetin administered intraperitoneally or orally

The extent and time course of induction of micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow were examined after administration of phenacetin as an insoluble suspension in olive oil by intraperitoneal injection (i.p.) or gastric intubation (p.o.) to 2 strains of mice, MS/Ae and CD-1, at doses up to 1200 mg/kg. The toxicity of phenacetin and the sensitivity of micronucleus induction differed in the 2 strains, but there was little difference in the extent of MNPCEs induced by the 2 administration routes.     

Rates of micronuclei induction in different mouse strains

It has previously been shown that the inbred mouse strain MS/Ae was more sensitive in the micronucleus test to several mutagenic agents than outbred mice. To elucidate the possible influence of inbreeding, several inbred strains including MS/Ae, AKR, BALB/c, C57 BR were compared to the two OF1 and NMRI outbred strains. The 3 mutagenic agents MNNG, MMC and MMS all induced a significantly higher number of micronuclei in the MS/Ae strain than in any of the other mouse strains. AKR was especially resistant to the alkylating agents MMS and MNNG. Hence, except for the MS/Ae mouse strain, no inbred strain showed a systematically higher sensitivity than the outbred strains for all of the 3 mutagenic agents used.     

Micronucleus test with procarbazine hydrochloride administered by intraperitoneal injection and oral gavage

The difference in effect of route of administration of procarbazine hydrochloride (PCZ) in the mouse was investigated in the micronucleus test. PCZ was administered by intraperitoneal injection (i.p.) and oral administration (p.o.) to 2 strains of male mice (MS/Ae and CD-1). On the basis of a small-scale acute toxicity test and a pilot micronucleus test, bone marrow preparations were prepared 24 h after the administration by the i.p. and p.o. routes of 50-400 mg/kg and 200-1600 mg/kg, respectively. The maximum incidence of polychromatic erythrocytes with micronuclei (MNPCEs) was somewhat higher after p.o. treatment in MS/Ae mice and the same with both routes in CD-1 mice. Thus, the clastogenicity of PCZ in mouse bone marrow was revealed by both routes.     

Micronucleus test with vincristine administered by intraperitoneal injection and oral gavage

The effects of vincristine sulfate (VINC) on micronucleus induction were studied in 2 strains of mice (MS/Ae: CD-1) following intraperitoneal (i.p.) or oral administration (p.o.) of the chemical. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the full-scale micronucleus test was performed with a sampling time of 24 h at doses of 0.063, 0.125, 0.25 and 0.5 mg/kg (i.p.) and 1.25, 2.5, 5.0 and 10 mg/kg (p.o.). The maximum frequency of micronucleated polychromatic erythrocytes was 7.15% in MS/Ae mice and 4.98% in CD-1 mice at 5.0 mg/kg p.o. in both cases. The maximum frequencies by the i.p. route (9.93% in MS/Ae mice; 11.68% in CD-1 mice) occurred at 0.25 mg/kg and 0.125 mg/kg, respectively. Although the doses showing a positive response were different between the 2 routes, VINC induced micronuclei very efficiently at all doses tested by both administration routes in both strains.     

Micronucleus test with 2-acetylaminofluorene by intraperitoneal injection and oral administration

The effect of route of administration on the outcome of the mouse micronucleus test was evaluated in 2 laboratories by administering 2-acetylaminofluorene (2-AAF) by intraperitoneal injection (i.p.) and oral gavage (p.o.) to 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus test, the full-scale experiment was performed with a 24-h sampling time at doses ranging from 75 to 600 mg/kg by both routes. The results indicated that 2-AAF induced micronucleated polychromatic erythrocytes (MNPCEs) at all doses tested by both routes. In the MS/Ae strain, higher doses were required by p.o. than by i.p. to reach a similar level of MNPCE incidence. On the other hand, similar responses were recorded by both administration routes with CD-1 mice. Since the LD50 for the p.o. route was higher than that for the i.p. route in both strains, the route-related difference with MS/Ae mice became small when the comparison between i.p. and p.o. was made on the basis of the LD50. Thus both i.p. and p.o. routes are acceptable in the micronucleus test of this chemical.     

Effect of route of administration in the micronucleus test with potassium bromate

The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice (MS/Ae and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 mg/kg in the i.p. test and of 37.5, 75, 150, and 300 mg/kg in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with MS/Ae mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.     

Administration-route-related differences in the micronucleus test with N-ethyl-N-nitrosourea

Administration-route-related differences in the micronucleus test were examined by giving N-ethyl-N-nitrosourea (ENU) to male mice of the MS/Ae and CD-1 strains by 2 different routes, intraperitoneally (i.p.) and orally (p.o.). The experiments consisted of 3 parts: (1) a simplified acute toxicity study, which gave LD50s of 490 (i.p.) and 840 mg/kg (p.o.) in MS/Ae and 640 (i.p.) and 960 mg/kg (p.o.) in CD-1 mice: (2) a pilot experiment for the full-scale micronucleus test to determine appropriate dosages and sampling time: and (3) the micronucleus test at doses of 12.5, 25, 50, and 100 mg/kg with a sampling time of 24 h. The results indicated that no route-related differences existed at the 2 lowest doses. At 50 mg/kg, markedly higher numbers of micronucleated polychromatic erythrocytes (MNPCEs) were induced in both mouse strains by the i.p. route. At 100 mg/kg, the difference between the routes decreased in strain CD-1 and even reversed in MS/Ae. Thus, route-related differences appeared to depend on the dose. Such differences became small, however, in both strains when the comparison was made on the basis of LD50 values.     

2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) is a weak in vivo clastogen as revealed by the micronucleus assay

The in vivo clastogenicity of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) was examined in the micronucleus test using peripheral blood from three mouse strains (ICR, CD-1, and MS/Ae) and bone marrow from one rat strain (Sprague-Dawley). Doses up to the maximum tolerated were tested. The chemical was given once, twice, thrice, or four times via either the i.p. or p.o. route. Under some conditions, ICR and CD-1 mice showed an increased frequency of micronucleated reticulocytes, but definite conclusions were difficult to draw because the increases were very slight. MS/Ae mice showed a markedly elevated micronucleated reticulocyte frequency after the double and triple ip treatments. Rats showed a slightly but statistically significantly increased frequency of micronucleated polychromatic erythrocytes after double i.p. treatments. These results indicate that AF-2 is a weak in vivo clastogen.     

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background

Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).

Methodology/Principal Findings

Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.

Conclusions/Significance

In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.     

Micronucleus test with cyclophosphamide administered by intraperitoneal injection and oral gavage

The effect of route of administration on the micronucleus test was examined in 2 laboratories: cyclophosphamide (CYP) was administered by intraperitoneal injection (i.p.) or oral gavage (p.o.) to 2 strains of mice. MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the final micronucleus test was performed with a 48-h sampling time at doses of 25-200 mg/kg i.p. and 50-400 mg/kg p.o. CYP via the i.p. route was more toxic and induced more micronucleated polychromatic erythrocytes (MNPCEs) in MS/Ae mice than in CD-1 mice. Administration-route-related differences were not distinctly shown in the MS/Ae strain. In CD-1, however, higher doses were required for the p.o. route than for the i.p. route to induce about equal amounts of clastogenic damage.     

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-epsilon, may slow the rates of cardiac contraction and relaxation (+dP/dt and -dP/dt). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser(43) and Ser(45) and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater -dP/dt than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/dt increased by 33% and -dP/dt increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.     

Diaziquone-induced micronuclei in cytochalasin B-blocked mouse peripheral blood lymphocytes

A mouse peripheral blood lymphocyte (PBL) micronucleus (MN) test was developed using a modification of the technique for assessing MN in human PBLs described by Fenech and Morley (1985). Male C57Bl/6 mice (5/dose) were injected i.p. with either 0, 2.5, 5.0, 7.5, or 10.0 mg diaziquone (AZQ)/kg. After 24 h the mice were bled by cardiac puncture, PBLs were isolated on a Ficoll-density gradient and then cultured in RPMI 1640 medium using 8 micrograms phytohemagglutinin/ml. In some cultures cytochalasin B (CYB) was added at 21 h during the medium change to block cytokinesis. In other cultures, CYB was omitted to compare the sensitivity of analyzing MN in binucleate versus unblocked mononucleate cells. All doses of AZQ yielded significant increases in MN-containing binucleated PBLs. The use of CYB in the mouse PBL MN test increased the sensitivity approximately 3-fold. The MN test in mouse PBLs should be useful in comparative cytogenetic studies of mice and humans.     

Micronucleus test with potassium chromate(VI) administered intraperitoneally and orally to mice

The effect of route of administration, intraperitoneal (i.p.) or oral gavage (p.o.), in the mouse micronucleus test was studied with K2CrO4 in 2 mouse strains (MS/Ae and CD-1). A simplified acute toxicity test to estimate the toxic dose levels of K2CrO4 showed that the LD50S were 50 mg/kg i.p. and 300 mg/kg p.o. for MS/Ae and 32 mg/kg i.p. and 180 mg/kg p.o. for CD-1. Based on results of a pilot micronucleus test to determine appropriate dose levels and the optimal sampling time, it was decided to sample bone marrow cells of both strains of mice 24 h after i.p. doses of 10-80 mg/kg and p.o. doses ranging from 20 to 320 mg/kg. K2CrO4 administered i.p. induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently in both strains. In contrast, when administered p.o. the chemical failed to induce MNPCEs. These results suggest that this difference between i.p. and p.o. routes is related to a difference of absorption or metabolic fate of chromate in vivo.     

Micronucleus test with methyl methanesulfonate administered by intraperitoneal injection and oral gavage

The effects of 2 routes of administration, intraperitoneal injection (i.p.) and oral gavage (p.o.), in the micronucleus test were evaluated using methyl methanesulfonate (MMS) and 2 strains of mice (MS/Ae and CD-1). A small-scale acute toxicity study and a pilot micronucleus experiment were carried out first. On the basis of the results obtained, a final micronucleus test was performed at doses of 20, 40, 80, and 160 mg/kg (i.p.) and 40, 80, 160, and 320 mg/kg (p.o.), with a 24-h sampling time. MMS induced micronucleated polychromatic erythrocytes (MNPCEs) in both routes in both mouse strains under the conditions used. At 40 and 80 mg/kg, MMS induced a higher number of MNPCEs by the i.p. route in both strains. A 160 mg/kg MMS dose induced higher numbers of MNPCEs by the p.o. route in MS/Ae mice. The route-related difference with MMS on the basis of mg/kg disappeared when the difference was determined on the basis of a ratio of the LD50. In practice, both i.p. and p.o. routes are acceptable as routes of administration in the micronucleus test using this chemical.     

Neuronal dystonin isoform 2 is a mediator of endoplasmic reticulum structure and function

Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum (dt) mice results in hereditary sensory neuropathy. Although loss of expression of neuronal dystonin isoforms (dystonin-a1/dystonin-a2) is sufficient to cause dt pathogenesis, the diverging function of each isoform and what pathological mechanisms are activated upon their loss remains unclear. Here we show that dt(27) mice manifest ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration through induction of a proapoptotic caspase cascade. dt sensory neurons display neurodegenerative pathologies, including Ca(2+) dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism of dt neuropathology and proposes a role for dystonin-a2 as a mediator of normal ER structure and function.     

Dystonia musculorum mutation and myosin heavy chain expression in skeletal and cardiac muscles.

This study evaluated the influence of dystonia musculorum (dt) mutation, characterized by spinocerebellar fibers degeneration, on cardiac and skeletal muscles: one respiratory (diaphragm, Dia), three masticatory (anterior temporalis, AT; masseter superficialis, MS; and anterior digastric, AD), one hindlimb (soleus, S), tongue (T), and one cardiac (ventricle, V). Body and muscle weight, muscle protein content, and myosin heavy chain (MHC) isoforms relative expression were then compared in dt mutant mice and in normal mice, according to sex. Male body and muscle weight was always greater than that of females, but there was no specific muscle difference in females. dt mutant mice showed a reduced whole body growth but no specific muscle atrophy, as well as a global decrease in muscle protein content that made muscles more fragile. dt mutation induced a global reduction of muscle protein concentration, whereas a general influence of sex could not be disclosed. Concerning MHC relative composition, all the muscles were fast-twitch: Dia, AT, MS, AD, S, and T expressed predominantly the fast type 2 MHC isoforms, whereas V contained only MHC alpha, also a fast MHC. Female muscles were slower than male muscles, except for S, which was faster. However, classification of muscles in terms of shortening velocity was very different in normal males and females. In other respects, dt mutant muscles were slower and consequently more fatigue resistant than normal, except for S, which became faster and less fatigue resistant. dt mutation exhibits then a specific effect on this continually active postural muscle. In the other muscles, global increased fatigue resistance could constitute an adaptive response to work requirements modifications linked to the muscle damage. It should be noted that a developmental MHC (neonatal) was present in female dt AD. Innervation, which influences muscle structure, is altered in dt mutant and could be another causal factor of the fast-to-slow MHC switches. It appears that dystonin, the dt gene product, is very important in maintaining the structural integrity of both cardiac and skeletal muscle and in its absence, the muscle becomes more fragile and is damaged by modified activity.     

Determination of cardiac contractility in awake unsedated mice with a fluid-filled catheter

Today, cardiac contractility in mice is exclusively measured under anesthesia or in sedated animals because the catheters available are too rigid to be used in awake mice. We therefore developed a new catheter (Pebax 03) to measure cardiac contractility in conscious mice. In this study, we evaluated the accuracy and utility of this new catheter for assessment of cardiac contractility in anesthetized and conscious mice. With the use of a balloon-pop test, the Pebax catheter with an inner diameter of 0.3 mm was found to exhibit a high natural frequency, a low damping coefficient, and a flat frequency of up to 50.5 +/- 0.6 Hz. Under anesthesia (0.5% or 1.0% halothane), no difference was found in heart rate (HR), left ventricular (LV) systolic pressure (LVSP), the maximum rates of LV pressure rise and fall (LV dP/dt(max) and LV dP/dt(min), respectively), ejection time (ET), and isovolumic relaxation time constant (tau) when measured with either the 1.4-Fr Millar or Pebax 03 catheter. However, when HR, LVSP, LV dP/dt(max), and LV dP/dt(min) were recorded with the Pebax catheter in awake mice, values were significantly higher, and ET and tau were lower, than under anesthesia, suggesting a major impact of anesthesia on these parameters. The Pebax catheter was also used in a normotensive one-renin gene mouse model of cardiac hypertrophy induced by DOCA and salt. In this model, DOCA-salt induced a severe decrease in cardiac contractility in the absence of changes in blood pressure. These data demonstrate that cardiac contractility can be measured very accurately in conscious mice. This new device can be of great help in the investigation of cardiac function in normal and genetically engineered mice.     

Overcoming unresponsiveness in experimental autoimmune encephalomyelitis (EAE) resistant mouse strains by adoptive transfer and antigenic challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2(b)) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated. The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 μg/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 μg/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.     

Cultivation of mosquito cell lines in serum-free media and their effects on dengue virus replication

Seven mosquito cell lines from five species (Aedes aegypti, Ae. albopictus, Ae. pseudoscutellaris, Culex tarsalis, and Toxorhynchites amboinensis) were adapted to three kinds of serum-free media (SEM), which were composed of equal volumes of tryptose phosphate broth and of either Leibovitz (L15) medium, Eagle's minimum essential medium, or Medium 199 with Hanks' salts. Population growth rates of the cells cultivated in the SMFs were generally slower than those of original cell cultures maintained in conventional media containing bovine sera. A karyological study showed a significant shift to heteroploidy in two of the four cell lines examined. Four SMF-adapted sublines were compared with parental cultures for replication of dengue viruses. Ae. aegypti RML-12, Ae. albopictus C6/36, Ae. pseudoscutellaris AP-61, and Tx. amboinensis TRA-171 demonstrated different levels of alteration in virus replication ranging from lower titers (as in Ae. albopictus C6/36) to comparable or higher titers (as in Ae. aegypti RML-12) when they were simultaneously inoculated with four dengue serotypes.