AIMTo evaluate the protective/ameliorative effects of vitamin E (vit E) on ethane dimethane sulfonate (EDS)-induced testicular toxicity in rats.

METHODSThe rats were assigned to eight groups, seven rats in each, and were injected intraperitoneally with vehicle, a single dose of ethane dimethane sulfonate (EDS) (75 mg/kg bodyweight), vit E (100 mg/kg bodyweight) or EDS + vit E for 3? days. Thereafter, the rats were weighed, anaesthetized with ether and killed by cervical dislocation. The left testis weights were recorded and the relative testis weights were calculated. The left testes were processed for routine paraffin embedding. Three right testes from each group were taken randomly and then processed for routine electron microscopy. Tissue sections were examined using light and electron microscopy, and were scored for histopathological changes.

RESULTSVit E coadministration did not prevent the bodyweight loss on days 3 and 7. However, vit E administration prevented the EDS-induced testicular-weight loss in rats that received vit E for 3 days but not 7 days. The relative testis weight was higher on day 3 (instead of on day 7) than other groups. Nevertheless, the testis histology was not markedly protected by vit E in the EDS-treated rats. Detailed microscopic assessment showed few Leydig cells and abundant fibroblast-like cells indicating only some protection.

CONCLUSIONVit E cotreatment showed partial protective effects on the testicular weight and testicular histology in rats that received EDS.

Animals ; Antitoxins ; pharmacology ; Male ; Mesylates ; toxicity ; Rats ; Rats, Sprague-Dawley ; Testis ; drug effects ; pathology ; Vitamin E ; pharmacology

The effect of four alkylating agents (MMS, EMs, DMN, DEM), under various con centrations on mouse bone marrow erythrocytes, were studied by means of the micronucleus test. The results obtained were as follows: 1) The lethal doses on mice were MMS = 130 mg/kg/bw, EMS = 300 mg/kg/bw, DMN = 50 mg/kg/bw and DEN = 70 mg/kg/bw. 2) Micronuclei were easily seen and in different controls the micronulei were found a little over 0.1%. 3) The dose-effect relationship was obtained. In the MMS and EMS treated groups, incidences of micronulei were 0.45 to 2.56% and 0.4 to 2.1% respectively. 4) In the DMN and DEN treated groups, incidences varied between 0.15 to 0.90 % and 0.2 to 1.02% respectively. 5) Four alkylating agents were compared and discussed with respect to micro nucleus production from chromosomal aberrations.
Animal ; Bone Marrow/ultrastructure ; Carcinogens/pharmacology* ; Cell Nucleus/drug effects* ; Chromosome Aberrations* ; Erythrocytes/ultrastructure ; Female ; Mesylates/pharmacology* ; Mice ; Mutagens/pharmacology ; Nitrosamines/pharmacology*

OBJECTIVETo study the effect of ethane dimethane sulfonate (EDS) injection on the volumes of different histological structures in the seminal vesicles of adult rats.

METHODSTwenty-seven male SD rats aged approximately 90 days were randomly divided into a control group (n = 14) and an EDS group (n = 13) to receive one intraperitoneal injection of normal saline and EDS (75 mg/kg bodyweight), respectively. At 7 and 12 days after treatment, the unilateral seminal vesicles were removed, methacrylate resin-embedded sections prepared and the total volumes of various structures in the seminal vesicles estimated using stereological methods.

RESULTSEDS treatment almost completely destroyed the Leydig cells in the testis, resulting in a drastic testosterone deficiency. The volume of the seminal vesicle (including the coagulating gland attached to the vesicle) was decreased by 53% in the 7 d EDS group (n = 6) in comparison with the 7 d control group (n = 7) ([138.2 +/- 12.9] vs [64.9 +/- 3.6] mm3, P < 0.01), but showed no significant difference between the 7 d and the 12 d EDS (n = 7) groups ([64.9 +/- 3.6] vs [55.4 +/- 7.7] mm3, P > 0.05). The total volumes of the glandular lumen, glandular epithelium, smooth muscular layer and adventitia were decreased by 96.7, 80.3, 57.6 and 67.0%, respectively, in the 12 d EDS group as compared with the 12 d control group (n = 7).

CONCLUSIONEDS induces drastic testosterone deficiency in adult rats, and significantly reduces the total volumes of the seminal vesicle lumen, glandular epithelium, smooth muscular layer and adventitia.

Animals ; Leydig Cells ; drug effects ; Male ; Mesylates ; pharmacology ; Organ Size ; drug effects ; Rats ; Rats, Sprague-Dawley ; Seminal Vesicles ; drug effects ; pathology ; Testis ; cytology ; drug effects ; pathology

To verify the differential expression of non-metastasis cell 3 (NME3) protein in HL-60 cells when they were induced to differentiate into monocyte and granulocyte like cells, and study its value in diagnosis of acute myeloid leukemia, all-trans retinoic acid (ATRA) and a new steroidal drug NSC67657 were employed to induce acute myeloid leukemia HL-60 cells into monocyte and granulocyte like cells. Then the cell differentiating direction was observed by chemical staining, the degree of differentiation was determined by surface antigen CD11b/CD14 detection, and the apoptosis was excluded by phosphatidylserine valgus analysis, by which cellular differentiating model was constructed. Furthermore, RT-PCR and Western blot were employed to verify the differentially expression of NME3 before and after differentiation of HL-60 cells. At last, samples from bone marrow nucleated cells of 26 patients with myeloid leukemia, which were diagnosed definitely by clinical doctors, and 5 normal people were chosen. Then the expressing trend of NME3 protein in these testing groups was analyzed by means of comparison. The results showed that ATRA (2 µmol/L for 5 d) and NSC67657 (10 µmol/L for 5 d) could induce HL-60 cells to differentiate into monocyte and granulocyte like cells above 90% without cell apoptosis. The expression of NME3 gene and protein were down-regulated by the inducers, which was accorded with the screening results that was got using proteomics technology in the former research. The expression of NME3 protein in bone marrow from acute myeloid leukemia patients was elevated significantly as compared to normal persons. It is concluded that the expression level of NME3 protein is down-regulated after cellular differentiation, according with the changing trend in leukemia patients, which imply that NME3 protein may be a potential biomarker for diagnosis of acute myeloid leukemia.
Adolescent ; Adult ; Aged ; Bone Marrow ; metabolism ; Case-Control Studies ; Child ; Child, Preschool ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Mesylates ; pharmacology ; Middle Aged ; NM23 Nucleoside Diphosphate Kinases ; metabolism ; Steroids ; pharmacology ; Tretinoin ; pharmacology ; Young Adult

OBJECTIVETo investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism.

METHODSThe HL-60 cells were treated with 5, 10 and 20 µmol/L XAV-939 (inhibitor of Wnt signalling pathway) for 3 days, and with 10, 20 and 30 mmol/L LiCl (activator of Wnt signalling pathway) for 1 day; the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot, respectively; the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry, moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined. Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 µmol/L for 3 days; the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control (culture mediam) group, simple NSC67657-treated group, NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed.

RESULTS20 µmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively, could significantly down- and up-regulate the expression of cyclinD1, TCF1 and c-Jun genes (P < 0.05) and proteins (P < 0.05); moreover, the number of CD10(+) HL-60 cells in these conditions was below 1%, no early apoptosis of HL-60 cells was found. In the simple NSC67657-treated groups, the expression of cyclinD1, TCF1 and c-Jun proteins was down-regulated (P < 0.05), and the percentage of CD14(+) HL-60 cells accounted for 62.13 ± 9.44; after the HL-60 cells were treated with XAV-939, the NSC67657 could more significantly down-regulate the expression of cyclinD1, TCF1 and c-Jun proteins and the percentage of CD14(+) HL-60 cell accounted for 84.17 ± 5.39%, as compared with simple NSC67657-treated group; as compared with blank controls group, the expression of cyclinD1, TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14(+) HL-60 cells decreased to 33.99 ± 8.37% in NSC67657 combined LiC1 streated group, but which were higher than those in simple NSC67657-treated group (P < 0.05).

CONCLUSION20 µmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins. The influence of XAV-939 and LiC1 on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.

Apoptosis ; Cell Differentiation ; Cyclin D1 ; metabolism ; Flow Cytometry ; HL-60 Cells ; Hepatocyte Nuclear Factor 1-alpha ; metabolism ; Humans ; Lipopolysaccharide Receptors ; metabolism ; Mesylates ; pharmacology ; Monocytes ; cytology ; Proto-Oncogene Proteins c-jun ; metabolism ; Steroids ; pharmacology ; Wnt Signaling Pathway

OBJECTIVETo figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.

METHODSThe differentiation of HL60 cells was induced by NSC67657, and the cell surface antigen CD14 expression was detected by flow cytometry. The gene and protein expressions of CCAAT enhancer binding protein alpha (C/EBPalpha) before and after the induction of cell differentiation were determined by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells, and its expression was verified. The effect of C/EBPalpha overexpression in HL60 cells was assessed by MTT assay, Wright's staining and flow cytometry before and after NSC67657 transfection.

RESULTSHL60 cells could be induced into monocytes by 10 micromol/L ATRA within 5 days, and the coverage of CD14 positive cells reached 93.9% after 5 days of drug treatment. The eukaryotic expressing vector was successfully constructed, and over 90% positive clones were obtained after screening by G418 and electrotransfection. The results of proliferative analysis, chemical staining, ultrastructural observation, and CD11b detection confirmed that HL60 cells could be induced into granulocytic differentiation by overexpression of C/EBPalpha protein. Moreover, in the drug treatment group, transfected cells could not be induced into monocytic differentiation, and their granulocytic differentiation was also inhibited.

CONCLUSIONThe monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPalpha protein-mediated signal transduction. However, the overexpression of CEBPalpha may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.

CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; CD11b Antigen ; metabolism ; Cell Differentiation ; drug effects ; Genetic Vectors ; Granulocytes ; cytology ; HL-60 Cells ; Humans ; Lipopolysaccharide Receptors ; metabolism ; Mesylates ; pharmacology ; Monocytes ; cytology ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; Steroids ; pharmacology ; Transfection