Micronucleus test on quinacrine dihydrochloride in mice: a comparison of dosage regimens

Micronucleus induction by quinacrine dihydrochloride (Q) was tested in CD-1 male mice by single, double, and triple oral treatment(s) with 50 or 250 mg/kg. The mice were killed 24 h after the last treatment. Femoral marrow cells were analyzed on slides stained with Giemsa and acridine orange. Both staining methods gave similar results. The frequencies of micronucleated polychromatic erythrocytes (MPE) were marginally increased, in comparison with a concurrent negative control group, only in the triple treatment group of 250 mg/kg. There were, however, no big differences in MPE frequencies among the treatment regimens.     

Comparison of single/multiple-dose protocols using triethylenemelamine and procarbazine hydrochloride for the mouse bone marrow micronucleus test

Treatment of mice with a single dose of either 4.8 mg/kg of triethylenemelamine (TEM) or 348 mg/kg of procarbazine hydrochloride (PC) induced higher frequencies of micronucleated polychromatic erythrocytes (MPE) after 48 h than after 24 h. The same observation was made when animals were treated with 1.6 or 8 mg/kg of TEM or 116 or 580 mg/kg of PC for 2 consecutive days (double-dose protocol). Surprisingly, the third dose of either 1.6 or 8 mg/kg of TEM caused lower MPE frequencies at the 72-h than at the 48-h sampling time. The observation that lower MPE frequencies after 72 h were also accompanied by reduced bone marrow toxicity might have reflected a drug-related adaptive reaction of the animals, for example the induction of detoxifying enzymes. Mean MPE frequencies as well as bone marrow toxicity were also slightly decreased after the third dose of either 116 or 580 mg/kg of PC, but statistical analysis showed no differences between the 48-h and the 72-h sampling times as regards the MPE frequencies and bone marrow toxicity. In addition to the high mean MPE frequency observed after 2 doses of 116 mg/kg of PC at the 48-h sampling time, a late increase in micronucleus induction was also seen after triple dosing at the 96-h sampling time. The present experiments with TEM and PC showed similar sensitivity for the multiple-dose assays when compared with the single-dose micronucleus test. In the case of the triple-dose assay, bone marrow toxicity proved to be a critical factor for appropriate dose selection. The computerized image analysis system was a convenient and time-saving tool for the automatic scoring of large quantities of cells for micronuclei as well as for the evaluation of bone marrow depression from the entire cell population analyzed for micronuclei.     

Beneficial Impact of CCL2 and CCL12 Neutralization on Experimental Malignant Pleural Effusion

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.     

Recombinant Human Endostatin Endostar Suppresses Angiogenesis and Lymphangiogenesis of Malignant Pleural Effusion in Mice

Background

Malignant pleural effusion (MPE) is a common complication of lung cancer. One widely used treatment for MPE is Endostar, a recombined humanized endostatin based treatment. However, the mechanism of this treatment is still unclear. The aim of this study was to investigate the effects of Endostar in mice with MPE.

Methods and Materials

Lewis lung carcinoma (LLC) cell line expressing enhanced green fluorescent protein (EGFP) was injected into pleural cavity to establish MPE mice model. Mice were randomly divided into four groups. High dose of Endostar (30 mg/kg), low dose of Endostar (8 mg/kg), normal saline, or Bevacizumab (5 mg/kg) was respectively injected into pleural cavity three times with 3-day interval in each group. Transverse computed tomography (CT) was performed to observe pleural fluid formation 14 days after LLC cells injection. Mice were anesthetized and sacrificed 3 days after final administration. The volume of pleural effusion n was measured using 1 ml syringe. Micro blood vessel density (MVD), Lymphatic micro vessel density (LMVD), the expression level of vascular endothelial growth factor A (VEGF-A) and VEGF-C were observed by immunohistochemistry (IHC) staining.

Results

The volume of pleural effusion as well as the number of pleural tumor foci, MVD and the expression of VEGF-A were significantly reduced in high dose of Endostar treat group. More importantly, LMVD and the expression of VEGF-C were markedly lower in treat group than those in the other three control groups.

Conclusion

Our work demonstrated that Endostar played an efficient anti-cancer role in MPE through its suppressive effect on angiogenesis and lymphangiogenesis, which provided a certain theoretical basis for the effectiveness of Endostar on the MPE treatment.     

Mitigation of caffeine-induced teratogenicity in mice by prior chronic caffeine ingestion.

Pregnant A/J female mice, which had drunk tap water or a 0.05% caffeine solution for 8-19 weeks after weaning, were each injected sc with 150 or 250 mg/kg caffeine once on day 13 of gestation. After 150 mg/kg caffeine the frequencies at term of fetal death, external malformation, and subcutaneous hematomas were significantly lower in the caffeine- than water-drinking group. After 250 mg/kg caffeine the frequency of fetal death but not of malformations and hematomas was lower in the group with caffeine pretreatment. These findings were explained by assuming that long-term ingestion of caffeine induced and increased rate of degradation of caffeine administered during pregnancy.     

The effect of diphenylhydantoin on the frequency of micronuclei in bone-marrow polychromatic erythrocytes of mice

Using the micronucleus test to evaluate the mutagenic effect of 5,5-diphenylhydantoin (DPH) on bone marrow polychromatic erythrocytes, male Balb-C mice were treated with the drug in single and multiple injection tests. A significant increase in the frequency of micronucleated polychromatic erythrocytes (MPE), P less than 0.05, was found when the mice received a single injection of DPH at doses of 0.5 and 1.0 mg/kg, and this frequency did not increase at higher doses. When mice were treated 3 times, at 24-h intervals, with 1.0 mg/kg of DPH, a significant increase in MPE was also observed (P less than 0.05) but this was lower than when they received a single injection of the same dose. A cytotoxic effect of NaOH, 0.1 N, which was used as solvent, was also observed either when alone or when DPH (1.0 mg/kg) was injected 3 times. This effect was comparable to the one produced by mitomycin C (MMC) at a dose of 0.5 mg/kg.     

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.     

Linear dose-response relationship of erythrocyte enzyme-activity mutations in offspring of ethylnitrosourea-treated mice

The specific activity of 10 erythrocyte enzymes was measured to detect gene mutations in F1 offspring of male mice treated with 3 different doses of ethylnitrosourea (ENU). After administration of ENU or of the solvent (controls), the (101/El X C3H/El)F1 hybrid males were mated to untreated T-stock females. No enzyme-activity mutant was found in 3610 F1 offspring of the control group. After treatment of postspermatogonial germ-cell stages, 1 mutant in 1125 F1 offspring of males treated with 160 mg ENU/kg body weight, and 2 mutants in 1319 F1 offspring of a 250-mg/kg group were observed. After treatment of spermatogonia, 9 enzyme-activity mutants in 4247 F1 offspring of males treated with 80 mg ENU/kg body weight, 15 mutants in 3396 F1 offspring of a 160-mg/kg group, and 9 mutants in 1402 F1 offspring of a 250-mg/kg group were detected. The mutation frequencies in spermatogonia were significantly different from that of the controls (P less than 0.01). The dose-response curve was found to be linear. The frequencies of enzyme-activity mutations are comparable to those of recessive specific-locus mutations determined in the same experiments. Enzyme-activity mutants with reduced activity as well as mutants with enhanced activity were found. Genetic and biochemical characterization of enzyme-activity mutants was routinely performed. In inter se crossings of heterozygotes, no offspring expressing a third phenotype other than the wild type and the heterozygote were found in approximately half of the mutation studies. The recovered mouse mutants might be used as animal models to study corresponding genetic diseases in humans.     

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.     

Carnitine and carnitine orotate affect the expression of the prolactin-releasing peptide gene

Carnitine is involved in fatty acid metabolism in mammals and is widely used as a nutritional supplement; carnitine orotate is a more absorbable form of carnitine. We investigated the effects of carnitine and carnitine orotate on mouse prolactin-releasing peptide (PrRP) mRNA expression. Twenty-four female mice were randomly divided into four groups of six; control mice were orally drenched with physiological saline solution (250 mg/kg body weight) and treatment mice were orally drenched with carnitine (250 mg/kg) or carnitine orotate (250 or 750 mg/kg), once a day, for 20 days from parturition. The carnitine or carnitine orotate was dissolved in saline solution before administration. The hypothalamus, pituitary and ovary were sampled on day 21 after parturition, and PrRP mRNA levels in these tissues were measured by semi-quantitative PCR, with glyceraldehyde 3-phosphate dehydrogenase as a control. Expression of PrRP in mice treated with carnitine and carnitine orotate was significantly increased in the ovary and significantly reduced in the pituitary gland. Compared with the control, hypothalamus PrRP mRNA increased significantly in the carnitine and low-dose carnitine orotate groups and decreased significantly in the high-dose carnitine orotate group. We conclude that carnitine and carnitine orotate regulate expression of PrRP in the pituitary gland and ovaries.     

Effects of chemically- and environmentally-induced hypothermia on micronucleus induction in rats

We investigated micronucleus induction in rats treated with chlorpromazine and reserpine, drugs that induce hypothermia. We administered chlorpromazine (31.3--250mg/kg) or reserpine (500--2000 mg/kg) intraperitoneally and measured temperature rectally. Chlorpromazine at 62.5-250mg/kg and reserpine at all doses significantly decreased rectal temperature, although the hypothermic response was weaker than previously reported in mice. Only chlorpromazine at 250mg/kg decreased rectal temperature transiently to <33 degrees C for 20h and induced a statistically significant increase in micronucleated polychromatic erythrocyte frequency. When rats treated with reserpine at 500mg/kg were exposed to an environmental temperature of 16 degrees C for 6, 12, or 24h to keep their body temperature under 33 degrees C, only the 24h treatment group significantly induced micronuclei. In addition, relatively large micronuclei (diameter of micronucleus> or = 1/4 diameter of cytoplasm) accounted for 33.0% of the induced micronuclei, suggesting that hypothermia affected the mitotic apparatus. The hypothermic response to chlorpromazine and reserpine was weaker in rats than in mice, and it was correspondingly more difficult to induce micronuclei in rats with those drugs.     

Inhibition of adriamycin-induced micronuclei by desferrioxamine in Swiss albino mice

Swiss albino male mice, 6–8 weeks old, were treated i.p. with different doses of desferrioxamine dissolved in water for 7 days. Some of the mice in each group were injected i.p. with adriamycin (15 mg/kg) and killed after 30 or 24, 48 and 72 h. The femoral cells of mice in different groups were collected and studied. Desferrioxamine treatment failed to affect the incidence of micronuclei at doses of 125–250 mg/kg/day. Pretreatment with desferrioxamine was found to provide significant protection against adriamycin-induced micronuclei without interfering with its cytotoxic potential.     

Susceptibility of a Brazilian wild rodent isolate of Schistosoma mansoni to praziquantel in mice

The therapeutic effects of praziquantel (PZQ) against a Schistosoma mansoni isolate derived from Nectomys squamipes (isolate R) and a susceptible isolate (BH) were analyzed in Swiss mice by fecal egg counting, adult worm reduction and oogram pattern. Infected mice were orally administrated with 62.5mg/kg (group 1), 125mg/kg (group 2), 250mg/kg (group 3) and 500mg/kg (group 4), each dose divided over 3 days (49, 50 and 51 days after infection). The data were analyzed using one-way analysis of variance (ANOVA). In regard to isolate R, no fecal eggs were observed with 250 mg/Kg and 500 mg/kg (p<0.05), whereas BH excretion reached zero with all doses. Mean worm burden reduction was significantly (p<0.05) higher at the two highest concentrations, regardless of isolate. At 62.5mg/kg, the percentage of immature eggs varied from 17% (isolate R) to 38% (isolate BH). At 125 mg/kg, the percentage of immature eggs varied from 20% (isolate R) to 16% (isolate BH). At 250 mg/kg, immature eggs dropped significantly to 1% (isolate R) and 4% (isolate BH). At 500 mg/kg, no immature eggs were found in isolate R, whereas in BH was 8%. No dosage significantly (p>0.05) affected the percentage of mature eggs, regardless of isolate. There was a large increase (p<0.001) in the percentages of dead eggs in all treated groups of 62% and 64% in groups 3 and 4, respectively (isolate R). The percentage of dead eggs rose from 34% (group 1) to 58% (group 3) in isolate BH. Although group 4 showed lowest increase in the percentage of dead eggs (46%), it was higher (p<0.001) compared to the 8% in the control. Our findings indicate that the wild isolate from N. squamipes is susceptible to PZQ.     

Altered response of strain of Schistosoma mansoni to oxamniquine and praziquantel

The susceptibility of a fourth generation Ouh strain (Paranapanema Valley, S?o Paulo, Brazil) of Schistosoma mansoni to oxamniquine (OXA) and praziquantel (PZQ) was studied. Ten groups of 13 female albino mice each were infected with 70 cercariae per animal. These mice were medicated orally on the 50th day after infection. Five groups were given OXA doses of 0, 100, 200, 300 and 400 mg/kg (single doses) and the rest were treated with PZQ doses of 0, 100, 200, and 250 mg/kg/5 days. Each group was sub-divided: 8 animals underwent perfusion after 15 days treatment, 5 mice followed up for oviposition and their feces were tested every 15 days for miracidia hatching. The efficacy of the OXA doses of 100 and 200 mg/kg was 66% and 91.4%, respectively and for the 100 mg/kg PZQ dose it was 90.1%. The follow-up groups with 100 and 200 mg/kg of OXA and PZQ, 100 and 150 mg/kg, showed that they re-established the oviposition after a period of 60 to 75 days of treatment. The ED50 was 69.6 mg/kg OXA and 39.4 mg/kg PZQ. The results show the tolerance of the Ouh strain to a dose of 100 mg with both drugs and they appoint the need for a dose review during the follow up of the oviposition and in monitoring phenomena in the field.     

Metabolic and pharmacokinetic studies of curcumin, demethoxycurcumin and bisdemethoxycurcumin in mice tumor after intragastric administration of nanoparticle formulations by liquid chromatography coupled with tandem mass spectrometry

This paper aims to investigate the metabolism and pharmacokinetics of curcumin, demethoxycurcumin and bisdemethoxycurcumin in mice tumor. To improve water solubility, nanoparticle formulations were prepared as curcuminoids-loaded solid lipid nanoparticles (curcuminoids-SLNs) and curcumin-loaded solid lipid nanoparticles (curcumin-SLNs). After intragastric administration to tumor-bearing ICR mice, the plasma and tumor samples were analyzed by liquid chromatography with ion trap mass spectrometry. We discovered that curcuminoids were mainly present as glucuronides in plasma, whereas in free form in tumor tissue. A validated LC/MS/MS method was established to determine the three free curcuminoids in tumor homogenate. Samples were separated on a Zorbax SB-C(18) column, eluted with acetonitrile-water (containing 0.1% formic acid), and detected by TSQ Quantum triple quadrupole mass spectrometer in selected reaction monitoring mode. The method showed good linearity (r(2)=0.997-0.999) over wide dynamic ranges (2-6000 ng/mL). Variations within- and between-batch never exceeded 11.2% and 13.4%, respectively. The extraction recovery rates ranged from 78.3% to 87.7%. The pharmacokinetics of curcuminoids in mice tumor fit two-compartment model and first order elimination. For curcumin-SLNs group, the dosing of 250 mg/kg of curcumin resulted in AUC((0-48 h)) of 2285 ngh/mL and C(max) of 209 ng/mL. For curcuminoids-SLNs group, the dosing equivalent to 138 mg/kg of curcumin resulted in higher tumor concentrations (AUC=2811 ngh/mL, C(max)=285 ng/mL). It appeared that co-existing curcuminoids improved the bioavailability of curcumin.     

N-Nitrosodi-n-propylamine induces organ specific mutagenesis with specific expression times in lacZ transgenic mice.

The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.     

大豆提取物（CKBN）对免疫低下小鼠免疫功能的影响

目的观察大豆提取物（CKBN）对免疫低下小鼠免疫功能的影响。方法腹腔注射环磷酰胺（cyclophosphamide,CTX）建立免疫功能低下小鼠模型,观察1、25、50、100 mg/kg剂量CKBN对免疫低下小鼠免疫功能的影响。结果25 mg/kg组的CKBN可显著增加免疫低下小鼠的脾指数;25、50、100 mg/kg组均能明显抑制环磷酰胺对小鼠外周血白细胞数量的影响,1 mg/kg组可显著提高单核细胞百分率,50 mg/kg组可显著提高中性粒细胞百分率;100 mg/kg组的IgG2a水平高于环磷酰胺组;1、25、50 mg/kg可显著提高腹腔巨噬细胞的吞噬功能;25 mg/kg组可显著提高NK细胞杀伤活性。结论CKBN能显著增强CTX造成的免疫低下小鼠的免疫功能。     

多色荧光原位杂交检测小鼠精子非整倍体

比多色荧光原位杂交技术评价了２－（４－噻唑）苯丙咪唑（ＴＢ）在雄性小鼠生殖形成过程中的非整倍体诱发效应。根据小鼠精子发育周期,以口饲法处理动物１１天,间隔２２天后取小鼠精子涂片,聚合应用８号,Ｘ及Ｙ染色体特异性ＤＮＡ探针进行多色ＦＩＳＨ,检测精子中出现的二倍体、双体、和缺本频率。结果发现：在２００ｍｇ／ｋｇ剂量组,上述３类异常精子频率均显著高于溶剂对照,其他２个剂量组的非整倍体精子频率与对照无显著     

The evaluation of a multiple dosing protocol for the mouse bone-marrow micronucleus assay using benzidine and 2,6-xylidine

Male ICR mice were treated with 1, 2 or 3 daily doses of either benzidine or 2,6-xylidine. Groups of 5 animals were sacrificed 24 h after the last dose and the bone marrow examined for micronuclei. Benzidine was given at dose levels of 40 and 200 mg/kg and 2,6-xylidine was given at dose levels of 75 and 375 mg/kg. These doses represent 10 and 50% of the respective median lethal doses. Benzidine produced a significant (p less than 0.001) dose related increase in the incidence of micronucleated polychromatic erythrocytes (MPE), while 2,6-xylidine had no effect on the frequency of micronucleated cells. Statistical analyses of the data indicated that the incidence of MPE was independent of the number of doses administered prior to bone marrow harvest.