[Capillary gas chromatographic analysis and mass spectrometric identification of modified DNA bases 5-methylcytosine and 8-hydroxyguanine].

The purpose of this paper was to establish a method for the determination of modified bases, 5-methylcytosine and 8-hydroxyguanine, in DNA by GC/FID. The experimental conditions were explored systematically for the quantitative analysis of these two modified bases, and the components were identified by GC/MS. The results showed that the variant components in DNA treated with Fenton's reaction can be derivatized and separated successfully. The relative molar reactive factors of 5-methylcytosine and 8-hydroxyguanine were 3.0 and 1.3, respectively. The sensitivity for them were 5.50 x 10(9) mV.s/g and 7.59 x 10(10) mV.s/g, respectively, while their detectable limits were 36.4 pg/s and 15.8 pg/s, respectively. The coefficients of variation for gas chromatograph were less than 5%, for derivatization, less than 6%, and for the whole analysis process, less than 20%.

Skeletal effects of constant and terminated use of risedronate on cortical bone in ovariectomized rats.

To study the skeletal effects of continual and terminated use of risedronate treatment on cortical bone in ovariectomized (Ovx) rats, we used risedronate (Ris), 5 microg x kg(-1), by subcutaneous injections, twice per week. The middle part of the tibial shafts (Tx) were processed undecalcified for quantitive bone histomorphometry. Cortical bone and the marrow areas of the tibial shaft did not change in either sham-Ovx or Ovx rats during the 150-day experimental period. Continued administration of Ris for 150 days decreased the marrow area and increased the percentage of cortical area compared with the matching sham and Ovx group. A decrease in bone formation indices in both periosteal and endocortical surfaces of Tx in sham-operated rats between the age of 5 and 8 months was seen. Ovariectomy increased the percentage of labeled perimeter in the periosteal area, and markedly increased the percentage of eroded perimeter in the endocortical surface compared with sham control groups in 81 and 150 days. Bone formation indices of Ris treatment were increased in periosteal surfaces, and percentages of eroded perimeter were decreased more in endocortical surfaces in 150 days than in the matching sham and Ovx groups. These data matched our static data, which showed a significantly increased percentage of cortical bone area and decreased percentage of marrow area. These bone gains were not maintained in the 90-day Ris withdrawal group. For cancellous bone, the 60-day Ris-treated high bone mass was maintained in the withdrawal group and not maintained in Ris continmuously treated group. These results indicate the effects of constant and terminated use of Ris in cortical bone were different from those in trabecular bone in the proximal tibial metaphysis.

Inhibitory effects of sodium quercetin monosulfate on pig platelet aggregation induced by thrombin.

AIM: To study the inhibitory effects of sodium quercetin monosulfate (SQMS) on pig platelet aggregation induced by thrombin. METHODS: Platelet aggregation was analyzed by turbidimetry. Cytosolic free calcium concentration ([Ca2+]i) was determined by Fura-2 fluorescence. Activity of protein kinase C (PKC) was assayed by incubating PKC with histone III S and [gamma-32 P] ATP. The cytoskeletal proteins were precipitated by Triton X-100 and separated by SDS-PAGE. RESULTS: SQMS inhibited the platelet aggregation induced by thrombin 500 U.L-1 with IC50 132 (50-347) mumol.L-1. SQMS inhibited Ca2+ influx in blood platelets induced by thrombin 500 U.L-1 in the presence of extracellular Ca2+ 1 mmol.L-1 with IC50 20 (9-46) mumol.L-1; SQMS inhibited the internal Ca2+ release in the absence of extracellular Ca2+. SQMS also decreased [Ca2+]i level in quiescent blood platelets. SQMS (10-160 mumol.L-1) inhibited the activity of cytosolic PKC from blood platelets in a concentration-dependent manner, but had no effect on membrane PKC. SQMS (20-80 mumol.L-1) inhibited the actin polymerization induced by thrombin 500 U.L-1 inblood platelets in a concentration-dependent manner. CONCLUSION: SQMS inhibited pig platelet aggregation induced by thrombin and its mechanism might be due to its inhibitions of Ca2+ influx, internal Ca2- release, PKC activity, and actin polymerization.

Effects of antitumor compounds isolated from Pteris semipinnata L on DNA topoisomerases and cell cycle of HL-60 cells.

AIM: To study the effect of the antitumor compounds 5F, 6F, and A from Pteris semipinnata L on the activities of DNA topoisomerases and cell cycle of HL-60 cells, and the synergism of compound 6F in combination with genistein in vitro. METHODS: DNA topoisomerases were isolated from HL-60 cell lines, and supercoiled pBR322 DNA was used as substrate to determine the activities of DNA topoisomerase I and II. Cell cycle was analyzed by flow cytometry (FCM). Cytotoxicity assay was tested by MTT method. RESULTS: Compounds 5F, 6F, and A inhibited the activities of DNA topoisomerase I and II. After exposure of the cells to compound 6F, an increase in cells in the S and G2/M phases and a decrease in cells in the G0/G1 phase of the cell cycle were observed. At low concentrations (57.8 and 115.6 nmol.L-1), compound 6F enhanced the cytotoxicity against HL-60 cell line in combination with genistein, q values were > 1.15. The enhancement times of 57.8 and 115.6 nmol.L-1 of 6F by genistein were 2.60 and 4.65, respectively. CONCLUSION: Compounds 5F, 6F, and A inhibited the activities of DNA topoisomerases of HL-60 cells. Compound 6F increased the number of cells in S and G2/M phases, decreased the population of G0/G1 phase cells, and enhanced the cytotoxicity of genistein, which had synergism with 6F in antitumor action.

Skeletal effects of constant and terminated use of sodium risedronate in ovariectomized rats.

AIM: To study the skeletal effects of constant and terminated use of sodium risedronate (Ris) treatment in the ovariectomized (Ova) rats. METHODS: Ris 5 micrograms.kg-1, s.c., twice a wk. The proximal tibial metaphysis (PTM) were processed undecalcified for quantitive bone histomorphometry. RESULTS: (1) Placebo-treated (normal saline) Ova rats were characterized by decreased trabecular area (TA) on d 60, d 81, and d 150 compared with aging controls, and bone resorption was over formation with high bone turnover. (2) Ova rats were treated with Ris for 60, 81, and 150 d (Ris-on) increased. (TA 217%, 108%, and 101%) respectively, vs Ova rats and depressed bone turnover indices to aging control level, but bone mass did not maintain at high level in 150-d group as in the early stage. (3) Ova rats were pretreated with Ris for 60 d and then terminated (Ris-on/off), followed by sequential sacrifice of rats on 21 and 90 d. Withdrawal on 21 d showed the same results as the match-age Ris-on group. Withdrawal on 90 d still maintained cancellous bone mass at a high level vs 150 d Ris-on groups (+26%) and aging control group (+27%). CONCLUSION: Regimen of Ris 60 d on then 90 d off prevented the development of osteoporosis in Ova rats.

[Effects of fructus cnidii coumarins compared with nilestriol on osteoporosis in ovariectomized rats].

AIM: To compare the total coumarins from dried fruits of Cnidium monnieri (TCCM) and nilestriol (Nil) against osteoporosis. METHODS: SD rats (40, female, 3-month-old) were randomly divided into basal control, age control, ovariectomized (Ova), Ova + TCCM 67 mg.kg-1, Ova + TCCM 200 mg.kg-1, 6 times a week, and Ova + Nil 1 mg.kg-1, i.g. once a week. After 12 wk, sections (20 microns) of proximal tibiae were examined histologically. RESULTS: Ova reduced markedly the trabecular bone mass due to bone resorption excessed bone formation (% Tb. Ar -59%). Treatment with TCCM 67 mg.kg-1 partly suppressed bone turnover, but did not inhibit bone loss in Ova rats (% Tb.Ar -43%). Treatment with TCCM 200 mg.kg-1 and Nil 1 mg.kg-1 increased the trabecular area (% Tb. Ar +100% and +274%). CONCLUSION: Nil was more potent than TCCM in protecting against osteoporosis in Ova rats via supression of bone turnover.

Effects of total coumarins of Fructus cnidii on skeleton of ovariectomized rats.

Total coumarins of Fructus Cnidii (TCFC), 5 g.kg-1 by intragastric gavage, 6 d/wk, x 7 wk, was effective for prevention of bone loss in ovariectomized (OVX) rats. In comparison to aging control rats, the proximal tibia of placebo-treated OVX rats were characterized by an increase in eroded perimeter (+298%), label perimeter (+77%), osteoid perimeter (+47%), mineral apposition rate (+32%) and bone formation rate (+130%). These changes indicated a high bone turnover in OVX rats leading to a rapid bone loss (-44%) in proximal tibial metaphysis. In contrast, the TCFC-treated OVX rats showed an increase of cancellous bone area (+41%) compared with placebo-treated OVX rats and decrease in all the above indices of bone turnover to near aging control levels except that of the osteoid area (+88%) which was higher than that in aging control, but mineralization lag time did not show significant changes. The results suggested that the TCFC inhibited the high bone turnover and reversed the bone loss at early menopausal stage.