OBJECTIVETo explore a flow cytometry (FCM)-based method for discriminating aneugen- or clastogen-induced micronuclei.

METHODSCells were stained with anti-CD71-FITC and PI, and the PI fluorescent signal intensity of micronucleated reticulocyte (MN-RET) in the peripheral blood of NIH mouse treated with COL or CP was detected by flow cytometry.

RESULTSThe ratio of the median of the intensity of MN-RET fluorescent signals to that of nucleated cell was low in the cyclophosphamide treated mouse, while the median was high in the colchicine treated mouse.

CONCLUSIONThe flow cytometry-based micronucleus assay can be used to discriminate primarily smaller MN induced by the clastogen exposure from the larger MN induced by an aneugen.

Animals ; Colchicine ; toxicity ; Cyclophosphamide ; toxicity ; Flow Cytometry ; methods ; Male ; Mice ; Micronuclei, Chromosome-Defective ; Mutagens ; toxicity ; Reticulocytes ; drug effects ; ultrastructure

OBJECTIVETo investigate the protective effect of epigallocatechin gallate (EGCG) on mouse sperm in vivo.

METHODSA total of 64 six-week-old male Kuming mice were randomly divided into eight groups of equal number to be treated with normal saline (negative control), Cyclophosphamide (CP) at 30 mg/kg (positive control), and CP followed by EGCG (experimental) at 20, 40, and 80 mg/kg, respectively, given every other day for 10 days. At 4 and 5 weeks after treatment, the bilateral testes of the mice were harvested for examination of sperm abnormality.

RESULTSEGCG did not increase the rate of CP-induced sperm abnormality in the mice, but reduced it instead with the prolonged time of treatment.

CONCLUSIONEGCG protects mouse sperm in vivo.

Animals ; Catechin ; analogs & derivatives ; pharmacology ; Cyclophosphamide ; toxicity ; Male ; Mice ; Mutagens ; toxicity ; Random Allocation ; Spermatozoa ; drug effects ; Time Factors

Animals ; Consciousness ; Cyclophosphamide ; toxicity ; Female ; Urinary Bladder, Overactive ; chemically induced ; physiopathology ; Urodynamics ; physiology

BACKGROUNDOveractive bladder (OAB) can be caused by many factors such as inflammation, bladder outlet obstruction, neurogenic factors. We performed an intraperitoneal (ip) injection of cyclophosphamide to induce cystitis in rats, which causes their detrusors to overact, to provide a valuable disease model for discussing OAB pathogenesis and to study effective curing methods.

METHODSFemale Sprague-Dawley rats were induced to form cystitis by cyclophosphamide (200 mg/kg, ip). The day after the injection, two catheters were inserted into each rat's bladder to study its urodynamics. The BL-410 model bio-function experimental system was used to monitor bladder pressure while the rats were conscious. Unstable detrusor contractions appear in the urine storage period as a standard to determine OAB, and the positive rate was calculated. Urodynamic parameters such as bladder basal pressure (BP), maximum voiding pressure (MVP), intercontraction interval (ICI), spontaneous activity (SA), maximum cystometric capacity (MCC), and bladder compliance (BC) were recorded in each group, and a light microscope was used to observe the pathological changes in the rat bladder tissue.

RESULTSThe detrusor instability rate of the model group was 83.33%. The MVP, MCC and BC of rats in the model group were lower than the control group (P < 0.01), and the BP, ICI and SA of the model group rats were higher than the control group (P < 0.01). The difference between the control group and the model group is statistically significant. The model group rats' bladder walls swelled and bled, the submucosa thickened and leukocyte infiltration became serious.

CONCLUSIONSAcute cystitis and OAB symptoms can be induced by ip injections of cyclophosphamide in rats. This can provide a valuable animal model to study OAB in human beings.

Animals ; Consciousness ; Cyclophosphamide ; toxicity ; Female ; Rats ; Rats, Sprague-Dawley ; Urinary Bladder, Overactive ; chemically induced ; physiopathology ; Urodynamics ; physiology

OBJECTIVETo observe the protective effects of vitamin E on the testicular injury by cyclophosphamide in mice, and the correlative mechanism.

METHODSFifty sexually mature male mice were randomly divided into five groups: the cyclophosphamide group (the CP group), the low-dose vitamin E group (the low-dose group), the middle-dose vitamin E group (the middle-dose group), the high-dose vitamin E group (the high-dose group), the matched control group (the control group). The first four groups were given cyclophosphamide by gavage at a dose of 5 mg/(kg x d). The low-dose group, the middle-dose group and the high-dose group were given vitamin E by subcutaneous injection at doses of 30 mg/(kg x d), 50 mg/(kg x d) , 70 mg/(kg x d) after 4 h of cyclophosphamide treatment. The control group was gavaged with equivalent normal saline. The treatment period for all groups was 28 days. The level of plasma FSH, LH, T and the activity of testicular SOD, GSHPx, CAT and the level of testicular MDA were detected. The histological structure and the ultrastructure of the testis were examined by light microscope and electron microscope.

RESULTSAs compared with the CP group, the plasma FSH, LH, T level and the SOD, GSHPx, CAT activity in the middle-dose group and the high-dose group were higher (P< 0.05, P< 0.01), MDA level significantly lower(P<0.01). The histological structure and the ultrastructure of the testis were in the normal range.

CONCLUSIONVitamin E has protective effects on the testicular injury by cyclophosphamide in mice. The possible mechanism of vitamin E may be its scavenging free radical and antioxidant effects, as well as it may have some stimulatory effects on gonadotrophin releasing of pituitary anterior lobe.

Animals ; Antioxidants ; pharmacology ; Cyclophosphamide ; antagonists & inhibitors ; toxicity ; Dose-Response Relationship, Drug ; Male ; Mice ; Mice, Inbred Strains ; Random Allocation ; Testis ; drug effects ; pathology ; Vitamin E ; pharmacology

OBJECTIVETo observe the effects of CMTM2 on cyclophosphamide (CP)-induced reproductive toxicity and the expression of steroidogenic acute regulatory (StAR) protein in the transgenic mouse model.

METHODSTwenty CMTM2 transgenic mice were equally divided into a CMTM2 + CP and a CMTM2 + NS group, the former intraperitoneally injected with CP at 50 mg per kg per d, while the latter with the equivalent dose of normal saline, both for 7 days. Another 20 wild C57BL/6J mice were randomly assigned to a WT + CP and a WT + NS group, treated the same way above. After 30 days, all the mice were sacrificed and their epididymides and testes removed for measurement of the serum testosterone level by radioimmunoassay, determination of sperm concentration and motility by light microscopy and detection of the expression of StAR by Western blot.

RESULTSThe levels of serum testosterone, sperm concentration and sperm motility were significantly decreased in the CMTM2 + CP group as compared with the CMTM2 + NS group ([42.98 +/- 3.25] nmol/L vs [46.74 +/- 3.38] nmol/L, [16.89 +/- 1.17 ] x 10(6)/ml vs [24.68 +/- 0.95 ] x 10(6)/ml, [72.75 +/- 1.25]% vs [85.14 +/- 1.12]%, P < 0.05), but remarkably less than in the WT + CP group ([37.97 +/- 4.17] nmol/L, [12.75 +/- 1.02] x 10(6)/ml, [50.52 +/- 1.37] %) (P < 0.05). However, the expression of StAR was significantly higher in the CMTM2 + CP than in the WT + CP group (1.16 +/- 0.07 vs 0.69 +/- 0.08, P < 0.05).

CONCLUSIONCMTM2 antagonizes cyclophosphamide-induced reproductive toxicity via regulating the expression of StAR, and hence plays a protective role in the reproductive system.

Animals ; Cyclophosphamide ; toxicity ; MARVEL Domain-Containing Proteins ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Repressor Proteins ; genetics ; Sperm Count ; Sperm Motility ; Testis ; drug effects ; metabolism

OBJECTIVETo study the effects of gonadotroph-releasing hormone (GnRH) agonist (GnRH-a) and GnRH antagonist (GnRH-ant) on cyclophosphamide (CTX)-induced follicle apoptosis in female rats.

METHODSThirty-six female Sprague- Dawley rats were randomized into 6 groups, namely normal saline (NS), CTX, GnRH-a+NS, GnRH-a+CTX, GnRH-ant+NS, and GnRH-ant+CTX groups. The rats were sacrificed between the first and second week after the treatments., and the follicle apoptosis was investigated using TUNEL assay and transmission electron microscopy.

RESULTSThe apoptosis rate of the granulose cells in the follicles in late development was significantly higher than that in early follicles, and the apoptosis rate of the oocytes and granulose cells in rats with CTX treatment was significantly higher than that in rats without CTX treatment (P<0.05). The apoptosis rate of the granulose cells in GnRH-a groups (ranging from 33.40 - or + 4.59 to 73.25 - or + 5.35) was significantly higher than that in GnRH-ant groups (27.46 - or + 4.52 to 49.38 - or + 5.02, P<0.05), but there was no significant difference in the oocytes of early follicles between GnRH-a groups (23.48 - or + 4.25 to 36.15 - or + 4.23) and GnRH-ant groups (21.47 - or + 3.81 to 34.04 - or + 5.54, P>0.05). Electron microscopy revealed characteristic apoptotic changes of the oocytes in early follicles and granulose cells in early and late follicles. The apoptotic changes were especially typical in the granulose cells showing the formation of the apoptotic bodies, and the oocytes only showed chromatin condensation and aggregation.

CONCLUSIONIn the rat mode, GnRH-a promotes while GnRH-ant suppressed follicle apoptosis induced by CTX. GnRH analogues regulates mainly granulose cell apoptosis, but have little effect on oocyte apoptosis.

Animals ; Apoptosis ; drug effects ; Cyclophosphamide ; toxicity ; Female ; Gonadotropin-Releasing Hormone ; analogs & derivatives ; antagonists & inhibitors ; Granulosa Cells ; pathology ; Oocytes ; pathology ; Ovarian Follicle ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo discover the characteristic changes of metabolic profiles in C57 mice with cyclophosphamide induced blood deficiency and the effect of Siwutang.

METHODAn integrated metabonomic study using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy has been applied to investigate the metabolic profiles of serum, aqueous and lipidic extracts of thoracic gland, spleen, bone marrow obtained from control, model group (intraperitoneal injection of cyclophosphamide at a dose of 250 mg x kg(-1)) and Siwutang treated model group. The NMR spectra were integrated in segments of 0.04 ppm and then analyzed by principal component analysis (PCA) using SIMCA-P software to visualize the similarities and differences in metabolic profiles between these groups.

RESULTPCA result showed conspicuous difference in the metabolic profiles between groups. Compared with the control group, the model group contained lower concentration of lactate, 3-hydroxybutyrate, choline, glucose, and higher concentration of VLDL/LDL, leucine/isoleucine in serum. Lower concentration of taurine choline, Fbeta:RCH2CH2CO, Epi-coprostanol and lactate were found in both in thoracic gland extracts and spleen extracts. And in spleen extracts, we also found the lower concentration of 3-HB. In the extracts of bone marrow, the lower concentration of lactate, choline, glucosee were observed. When they were dosed with Siwutang 10 g x kg(-1) x d(-1) for 7 days, the effects above-mentioned were reversed.

CONCLUSIONThe injury established by injecting CTX is a kind of proper model to develop further metabonomics researches. The damage of mitochondria, disorder of energy metabolism and osmoregulation are observed in cyclophosphamide caused blood deficiency model by NMR-based-metabonomics method, and the Siwutang can improve these effects.

Anemia ; drug therapy ; metabolism ; Animals ; Cyclophosphamide ; toxicity ; Drugs, Chinese Herbal ; therapeutic use ; Magnetic Resonance Spectroscopy ; methods ; Medicine, Chinese Traditional ; Metabolomics ; Mice ; Mice, Inbred C57BL ; Paeonia ; Principal Component Analysis

Even though uroplakins (UPs) are believed to serve a strong protective barrier against toxic materials, cyclophosphamide (CP) causes extensive cystitis. We investigated the expression of UPs in the urothelium in CP induced mouse cystitis. A total of 27 ICR female mice received a single intraperitoneal injection of 200 mg CP/kg. Nine CP-treated mice and 6 controls were sequentially killed at 12, 24, and 72 hr post injection. Extensive cystitis and an increased vesical weight were seen. These all peaked within 12 hr post injection and they tended to decrease thereafter. The level of all the UPs mRNA, the protein expressions of UP II and III on immunoblotting study, and the expression of UP III on immunolocalization study were maximally suppressed within 12 hr; this partially recovered at 24 hr, and this completely recovered at 72 hr post CP injection. In conclusion, CP reduced the expression of UPs. The reduction of the UPs mRNA and protein was time dependent, and this peaked within 12 hr after CP injection. However, the damage was rapidly repaired within 24 hr. This study demonstrates a dynamic process, an extensive reduction and rapid recovery, for the UPs expression of the mouse urinary bladder after CP injection.
Animals ; Cyclophosphamide/*toxicity ; Cystitis/chemically induced/*metabolism/pathology ; Female ; Immunosuppressive Agents/*toxicity ; Membrane Glycoproteins/*metabolism ; Membrane Proteins/*metabolism ; Mice ; Mice, Inbred ICR ; RNA, Messenger/metabolism ; Time Factors ; Urinary Bladder/*metabolism

OBJECTIVETo study the effect of Morinda officinalis (MO) extract on cytoxan (CTX) -impaired spermatogenesis of adult male SD rats.

METHODSWe randomly divided 56 adult male SD rats into seven groups of equal number: blank control, CTX model, CTX + NS, CTX + 10 g/kg MO, CTX + 20 g/kg MO, CTX + 30 g/kg MO, and CTX + 40 g/kg MO. We made the models of impaired spermatogenesis in the SD rats by intraperitoneal injection of CTX and treated the animal models by intragastric administration of MO at the concentrations of 10, 20, 30, and 40 g per kg per d, respectively. After two weeks of medication, we observed the changes in the body weight, testicular and epididymal indexes, and microstructure of the testis tissue, measured the mean seminiferous tubule diameter (MSTD) , and obtained testicular biopsy scores (TBS) in different groups, followed by comparative analyses.

RESULTSAfter treatment, the CTX + NS group showed no remarkable differences in the body weight ([234.83 ± 28.77] g) and epididymal index (2.71 ± 0.34) from those of the four CTX + MO groups, but exhibited a significantly lower testicular index ([12.15 ± 1.04] g) than those in the CTX + 20 g/kg MO ([13.71 ± 0.97] g), CTX + 30 g/kg MO, ([13.30 ± 0.29] g), and CTX + 40 g/kg MO group ([13.48 ± 0.51] g) (P < 0.05). Light microscopy revealed obvious pathological changes of the testis tissue in the CTX + NS group and significantly ameliorated structures of the seminiferous tubules in the CTX + MO 10, 20, 30, and 40 g/kg groups, with the MSTD of (204.78 ± 11.03), (216.55 ± 10.93), (218.03 ± 11.23), and (218.59 ± 14.06) μm, respectively, and the TBS of 9.03 ± 0.39, 9.69 ± 0.26, 9.83 ± 0.18, and 9.89 ± 0.11, respectively, all significantly higher than (189.74 ± 8.55) μm and 5.95 ± 1.21 in the CTX + NS group (P < 0.05). The efficacy of MO extract was increased in a concentration-dependent manner.

CONCLUSIONMorinda officinalis extract can repair cytoxan-induced damage to rat spermatogenesis, with may achieve the best effect at the concentrations of 30 and 40 g per kg per d.

Animals ; Body Weight ; drug effects ; Cyclophosphamide ; toxicity ; Epididymis ; drug effects ; Male ; Morinda ; chemistry ; Mutagens ; toxicity ; Plant Extracts ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Seminiferous Tubules ; drug effects ; pathology ; Spermatogenesis ; drug effects ; Testis ; drug effects ; ultrastructure