Comparative developmental toxicity of cationic and neutral rhodamines in mice

Rhodamines 123 and 6G (Rh 123 and Rh 6G) are cationic fluorescent dyes that inhibit oxidative phosphorylation following their selective accumulation within mitochondria. Neutral rhodamines (e.g., Rh 116 and Rh B) do not share these properties. To determine if cationic and neutral rhodamines differ in their effect on mammalian development, pregnant CD-1 mice were injected i.p. with Rh 123, Rh B, or Rh 116 at doses of 15 mg/kg/day. The rhodamines were given alone or in combination with 500 mg/kg/day 2-deoxy-D-glucose (2-DOG), an inhibitor of glycolysis, daily on gestation days 7-10 (copulation plug = day 1). Additional pregnant mice were similarly treated with Rh 6G at a dose of 0.5 mg/kg/day. Controls were given saline equimolar to the dose of 2-DOG. Treatment with Rh 6G, alone or in combination with 2-DOG, significantly increased the incidences of prenatal mortality (17% and 35%, respectively) when compared with the control incidence (6%). Treatment with Rh 123 or Rh 6G, alone or with 2-DOG, inhibited fetal growth. Treatment with the neutral rhodamines had little effect on prenatal survival or growth. Exposure to Rh 6G, with or without 2-DOG, was associated with high incidences of gross malformations (41% and 61%, respectively). Rh 116 or Rh B, with or without 2-DOG, and Rh 123 alone were not associated with statistically significant teratogenic effects, but results of the latter treatment were suggestive of such an effect (9.1% grossly malformed fetuses vs. 0% for controls). The incidences of skeletal malformations were significantly increased in the test groups given Rh 6G + 2-DOG, Rh 123 + 2-DOG, or Rh 6G alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioprotective effect of pyrazolone derivatives]

The intraperitoneal injection of analgin (1000 mg/kg), antipurine (400 mg/kg), amidopyrine (100 mg/kg) 3 hours before the irradiation of mice in a dose of 800 R led to survival of 30 to 45% of the animals (against 12.5% in control) and to increase in the average duration of life of the animals that perished. 80-95% of mice survived the period of "intestinal deaths" (the 7th day after the irradiation) after combined prophylactic use of purasolone derivatives and cystamine before the irradiation of these animals in a dose of 1050 R. The radioprotective effect of pyrasolone derivatives given in therapeutic doses was less pronounced.     

氯胺酮对小鼠Ⅲ度烧伤死亡率和瘢痕形成的作用

目的观察氯胺酮对小鼠Ⅲ度烧伤死亡率和疤痕形成的作用,为临床应用提供实验依据。方法小鼠背部用95%酒精造成Ⅲ度火焰烧伤,烧伤面积7cm2,烧伤时间30s;氯胺酮用量10～20mg/kg,分别于烧伤后或烧伤前后腹腔注射,对照组注射等量生理盐水,通过烧伤小鼠早期死亡率和烧伤后期瘢痕面积观察其作用。结果 BALB/c和C57BL/6小鼠烧伤后立即、4h、24h腹腔注射氯胺酮20mg/kg,小鼠死亡率分别为42.8%和100%,与对照组无明显差别（P〉0.05）;C57BL/6小鼠烧伤前10min和烧伤后4h、24h注射氯胺酮10mg/kg,小鼠死亡率为100%,与对照组也无差别。烧伤时间减为20s,于烧伤烧伤后立即和24h注射氯胺酮20mg/kg,35d后测定瘢痕面积,治疗组明显小于对照组（P〈0.05）。结论氯胺酮无明显降低小鼠烧伤早期死亡率的效果,但有减少烧伤瘢痕面积的作用,可能与氯胺酮抗炎作用有关。     

Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.     

Effect of corticosteroids on arachidonate induced mortality in male and female mice

Sodium arachidonate (50 mg/kg) given intravenously to male and female mice induces pulmonary emboli followed by respiratory distress and cyanosis. Female mice are significantly more resistant to this treatment than male mice. Cortisone pretreatment for four days to intact mice (10 mg/kg/day/4 days) had a significant protective effect in both males and females against arachidonate toxicity, eliminating the sex difference previously observed. Adrenalectomy four days before arachidonate infusion increased the sensitivity to SA and resulted in 100% mortality in both sexes. Pretreatment of adrenalized animals with cortisone significantly reduced to some degree the mortality rate in both sexes. Castration of male and female mice three weeks before adrenalectomy did not affect the mortality rate seen following adrenalectomy alone. In conclusion, exogenous cortical steroids augment the resistance of even intact mice and are absolutely necessary for survival in adrenalectomized animals. The observed sex differences in untreated intact animals is not seen after treatment with cortisone or adrenalectomy.     

精原干细胞移植受体鼠模型的建立

目的 通过对4周龄昆明白小鼠腹腔内单次注射白消安来制作精原干细胞移植受体鼠模型。方法 将实验动物分为4组,A、B、C组注射白消安的剂量分别是30 mg/kg4、0 mg/kg5、0 mg/kg体重,D组为对照组。注射后每天记录小鼠的存活情况,注射白消安后的20 d3、0 d4、0 d称量睾丸重量、测定血常规、制作并观察睾丸组织学切片、统计曲细精管的中空率。结果 A、B、C、D组的死亡率分别是25.00%3、1.58%、80.00%、0.00%,注射白消安后30 d各组小鼠曲细精管中空率分别是45.25%、75.25%、1.50%、0.00%,白细胞、红细胞、血小板数量等血常规指标均恢复正常。结论 腹腔内单次注射30 mg/kg或40 mg/kg剂量的白消安,死亡率较低（25.00%、31.58%）,30 d后曲细精管中空率较高（45.25%7、5.25%）、血常规指标恢复正常,适合做精原干细胞移植受体。     

Hemopoietic function after use of IL-1 with chemotherapy or irradiation

IL-1 has putative chemo- and radioprotective properties, but its effects on primitive hemopoietic stem cell (PHSC) and early multilineage precursor function when given with these modalities is unknown. C57BL6/J (B6) mice, given IL-1 20 h before cyclophosphamide (200 mg/kg for four biweekly doses) or before irradiation (500 cGy), were sacrificed after 4 wk. Their marrow was used as donor cells, and that from B6-Hbb(dGpi1a) (B6-GPI) mice was used as competitor cells in competitive repopulation. Percentages of B6 cells were measured at 30 and 150 days. Stem cell numbers were estimated using binomial statistics. IL-1 alone did not affect stem cell function. As expected, significant declines in early multilineage precursor and PHSC function occurred with chemotherapy and radiation alone. IL-1 with chemotherapy led to exacerbation of these losses in function and numbers (p < 0.05). A similar reduction in function occurred using IL-1 before irradiation. In summary, IL-1 with chemotherapy or radiation worsened chemotherapy- and radiation-induced functional damage to PHSC and other hemopoietic precursors, suggesting that improvements in survival do not necessarily translate into preservation of hemopoietic function.     

Trehalose dimycolate enhances survival of fission neutron-irradiated mice and Klebsiella pneumoniae-challenged irradiated mice

The survival of B6D2F1 female mice exposed to lethal doses of fission neutron radiation is increased when trehalose dimycolate (TDM) preparations are given either 1 h after exposure or 1 day before exposure to radiation. TDM in an emulsion of squalene, Tween 80, and saline was the most effective formulation for increasing the 30-day survival of mice when given 1 day before (90%) or 1 h after (88%) exposure to radiation. An aqueous suspension of a synthetic analog of TDM was less effective at increasing 30-day survival (60%) when given 1 day prior to radiation exposure and not effective when given 1 h after radiation. Mice receiving a sublethal dose (3.5 Gy) of fission neutron radiation and either the TDM emulsion or synthetic TDM 1 h after irradiation were substantially more resistant to challenge with 10, 100, 1000, or 5000 times the LD50/30 dose of Klebsiella pneumoniae than untreated mice.     

Protection by interleukin 1 against lung toxicity caused by cyclophosphamide and irradiation

Interleukin 1 has been shown to provide protection against the toxic effects of cyclophosphamide given in combination with localized irradiation of the lung. A single dose of 15 micrograms/kg interleukin 1 was given 24 h before cyclophosphamide (25-125 mg/kg) which was followed 1 day later by five daily exposures of 4.5 or 5.0 Gy, localized to the lung. Interleukin 1 significantly reduced early measurements of breathing rate for mice treated with high cyclophosphamide doses and irradiation, and at cyclophosphamide doses above 50 mg/kg, there was a significant reduction in the lethality from the combined treatment. At lower levels of cyclophosphamide, however, there seemed to be no effect.     

Interference of Mycotoxins with Prenatal Development of the Mouse

The prenatal effects of mycotoxins were investigated in GBA mice given by stomach tube a single dose of either aflatoxin B1 (4 mg/kg), ochratoxin A (8 mg/kg) or zearalenone (20 mg/kg) on pregnancy day 8 or 9. Aflatoxin caused foetal anomalies (exencephaly, open eyes, and protrusion of intestines) after exposure on gestation day 8 but not on day 9. The effects (increased prenatal mortality, reduced foetal growth, and a wide variety of malformations) caused by ochratoxin were much more severe and occurred after treatment on either of the 2 days of gestation. Among the spectrum of malformations, predominantly involving the craniofacial complex and the axial skeleton, the most striking was the total aplasia/dysplasia of the upper facial structures. These defects were always accompanied by exencephaly and anophthalmia. Zearalenone caused no effects. It is concluded that of the 3 mycotoxins screened with the technique used, ochratoxin is the most potent teratogen in mice.     

Elimination of allogeneic inhibition of hematopoietic stem cells by treatment of the recipient mice with cyclophosphane]

The experiments demonstrated that pretreatment of lethally irradiated recipient (CBA X C57BL/6) F1hybrid mice with cyclophosphamide (200 mg/kg of body weight) on day before the bone marrow transplantation (4 hours after the irradiation) suppressed the allogeneic inhibition of hematopoietic stem cells to 24% (while the inhibition in the untreated animals was 92.5%). It is suggested that cyclophosphamide acted on the recipient's radioresistant lymphoid cells effecting the allogeneic inhibition of stem cells.     

In vivo radioprotection by alpha-TMG: preliminary studies

alpha-TMG is a novel water-soluble derivative of Vitamin E that has shown excellent antioxidant activity. The parent compound has demonstrated protection against radiation induced chromosomal damage in vivo. Hence, the preliminary experiments to determine the radioprotective activity of alpha-TMG were carried out in adult Swiss albino mice. Acute toxicity of the drug was studied taking 24h, 72 h and 30 day mortality after a single intraperitoneal injection of 500-2000 mg/kg body weight of the drug. The drug LD(50) for 24h and 72 h/30 day survival were found to be 1120 and 1000 mg/kg body weight, respectively. The optimum time of drug administration and drug dose-dependent effect on in vivo radiation protection of bone marrow chromosomes was studied in mice. Injection of 600 mg/kg of the drug 15 min before or within 5, 15 or 30min after 3Gy whole body gamma radiation resulted in a significant decrease in the aberrant metaphases percent at 24h post-irradiation; the maximum effect was seen when the drug was given immediately after irradiation. Injection of 200-800 mg/kg TMG within 5 min of irradiation with 3 Gy produced a significant dose-dependent reduction in the radiation induced percent aberrant metaphases and in the frequency of micronucleated erythrocytes at 24h after exposure, with a corresponding decrease in the different types of aberrations. The optimum dose for protection without drug toxicity was 600 mg/kg body weight. At this dose, TMG produced 70 and >60% reduction in the radiation induced percent aberrant metaphases and micronucleated erythrocytes, respectively. The high water solubility and effectiveness when administered post-irradiation favor TMG as a likely candidate for protection in case of accidental exposures.     

In vivo radioprotective effect of Moringa oleifera leaves

Radioprotective property of Moringa oleifera leaves was investigated in healthy adult Swiss albino mice. Animals were injected (ip) with 150 mg/kg body weight of 50% methanolic extract (ME) of M. oleifera leaves, as a single dose, or in 5 daily fractions of 30 mg/kg each, and exposed to whole body gamma irradiation (RT, 4 Gy) 1 hr later. Five animals from each group were sacrificed at 1, 2 and 7 days after treatment. Bone marrow protection was studied by scoring aberrations in metaphase chromosomes and micronucleus induction in polychromatic erythrocytes and normochromatic erythrocytes. Pretreatment with a single dose of 150 mg/kg ME significantly reduced the percent aberrant cells to 2/3rd that of RT alone group on day 1 and brought the values to normal range by day 7 post-irradiation. A similar effect was also seen for the micronucleated cells. Fractionated administration of ME (30 mg/kg x 5) gave a higher protection than that given by the same dose administered as a single treatment. ME also inhibited the Fenton reaction-generated free radical activity in vitro in a concentration dependent manner. These results demonstrate that pretreatment with the methanolic leaf extract of M. oleifera confers significant radiation protection to the bone marrow chromosomes in mice and this may lead to the higher 30 day survival after lethal whole body irradiation.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

Sensitivity of mice to systemic hyperthermia: effect of the transplantation of ascites tumor cells modified by preheating, and by an injection of CDDP or interferon

Effects of preheating and injection of cis-DDP (CDDP) or interferon on tumor-induced sensitization to systemic hyperthermia (SH) was investigated in mice. LD50 of SH at 42.0 +/- 0.2 degrees C (core body temperature) was 43 min in normal mice and 8 min in mice which were i.p. transplanted with FMA3 cells at a dose of 10(5) one day before. In mice which had received the SH for 10 min one hour before, one hour after or one day before the transplantation, LD50 of the SH one day after the transplantation was 41, 35 and 22 min, respectively. An injection of CDDP given i.p. at a dose of 4 mg/kg one day after the transplantation, which was effective to kill about 99% of the tumor cells, did not change the course of thermosensitization after the transplantation. An i.p. injection of mouse interferon did not change the thermosensitivity of normal mice, but greatly suppressed the thermosensitizing effect of tumor cells when it was given one day before the transplantation.     

Photoinduced antitumour effect of hypericin can be enhanced by fractionated dosing

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80mm3 the mice were intraperitoneally injected with hypericin, either in a single dose (5mg/kg; 1 or 6h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70mW/cm2, 168 J/cm2). All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05). Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.     

Effects of X-irradiation and adriamycin on quiescent and proliferating cells of the seminal vesicle in the castrated mouse.

The sensitivity of resting and proliferating cells of the seminal vesicle to X-irradiation and adriamycin has been investigated. Stimulation with testosterone propionate (250 micrograms/day) was started 11 days after castration in BALB/c mice. X-rays (2.5-7.5 Gy total body irradiation) and intraperitoneal injections of adriamycin (4-16 mg/kg body weight) were administered at various times before or after induction of proliferation by testosterone injection. The DNA contents and the weights of the seminal vesicles were determined at 4 days after the start of stimulation. A Do for X-rays of about 10 Gy was found for the seminal vesicle epithelium. For both X-irradiation and adriamycin no significant differences in sensitivity were observed between quiescent (Go) and proliferating (G1; S) seminal vesicle cells.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Cochleates: New Lipid-Based Drug Delivery System

Abstract

Cochleates are a lipid-based tailored drug delivery system formed by the precipitation of a negatively charged lipid and a cation, for example phosphatidylserine and calcium. Hydrophobic, amphiphilic, negatively or positively charged moieties are suitable candidates to be delivered via cochleates. Various procedures have been developed allowing the control of cochleate particle size, including the trapping and hydrogel methods, which use either a direct addition or a slow diffusion of calcium into the negatively charged liposome/drug suspension. The efficacy of cochleates to encapsulate and deliver drugs was evaluated using amphotericin B as a model. Amphotericin B cochleates (CAMB) were compared to Fungizone® and AmBisome®, two commercially available AmB products. Parenterally, CAMB was given IP to ICR mice infected with Candida albicans. 100% survival was observed with low doses of CAMB (0.5 mg/kg/day, 10 days) compared to 60% for Fungizone, at the same dose. Tissue burden studies were conducted in parallel. Mice were treated daily from day 1 to day 7 post challenge and tissue burden assessed at day 8. In the kidneys, all three formulations were comparable in reducing colony counts. In the spleen, CAMB at 10 mg/kg/day was comparable to AmBisome given IV at the same dose. At 1 mg/kg/day, CAMB was more potent than Fungizone and AmBisome. Oral administration of CAMB in C57BL/6 mice, at 10 mg/kg results in high levels of AmB in target tissues. Multiple daily doses (10) showed accumulation of AmB in key tissues (liver, lungs, spleen, and kidneys) and AmB tissue concentrations are raised to therapeutic levels. Orally administered CAMB are highly effective against fungal infections in mice at very low doses. Balb/C mice were infected with Candida albicans and were given oral CAMB as a daily dose for 15 days. Comparison was done to AmBisome given orally at 10 mg/kg and Fungizone IP. 100% survival was obtained with CAMB at doses as low as 0.5 mg/kg/day (15 days). CAMB eradicate Candida from lungs when given at 2.5 mg/kg/day and was comparable to Fungizone given IP at almost the same dose (2 mg/kg/day). The comparison between CAMB and AmBisome shows that oral CAMB is 10 times more effective than oral AmBisome in reducing colony counts in both kidneys and lungs. Orally administered CAMB were non-toxic even at the highest dose of 50 mg/kg/day (14 days). This was demontrated by 100% survival of the animals and normal histopathology analysis. No lesions in the kidneys, GI tract, lungs, liver and spleen was observed despite the substantial amount of AmB in these organs. AmB cochleate promise to be a safe, broad spectrum, effective and orally available, antifungal formulation.