Genotoxic effects of nanosized and fine TiO2.

The in-vitro genotoxicity of nanosized TiO(2) rutile and anatase was assessed in comparison with fine TiO(2) rutile in human bronchial epithelial BEAS 2B cells using the single-cell gel electrophoresis (comet) assay and the cytokinesis-block micronucleus test. BEAS 2B cells were exposed to eight doses (1-100 microg/cm(2)) of titanium(IV) oxide nanosized rutile (>95%, <5% amorphous SiO(2) coating; 10 x 40 nm), nanosized anatase (99.7%; <25 nm), or fine rutile (99.9%; <5 microm) for 24, 48, and 72 h. Fine rutile reduced cell viability at lower doses than nanosized anatase, which was more cytotoxic than nanosized rutile. In the comet assay, nanosized anatase and fine rutile induced DNA damage at several doses with all treatment times. Dose-dependent effects were seen after the 48- and 72-h treatments with nanosized anatase and after the 24-, 48- (in one out of two experiments), and 72-h treatments (one experiment) with fine rutile. The lowest doses inducing DNA damage were 1 microg/cm(2) for fine rutile and 10 microg/cm( 2) for nanosized anatase. Nanosized rutile showed a significant induction in DNA damage only at 80 microg/cm(2) in the 24-h treatment and at 80 and 100 microg/ cm(2) in the 72-h treatment (with a dose-dependent effect). Only nanosized anatase could elevate the frequency of micronucleated BEAS 2B cells, producing a significant increase at 10 and 60 microg/cm( 2) after the 72-h treatment (no dose-dependency). At increasing doses of all the particles, MN analysis became difficult due to the presence of TiO(2) on the microscopic slides. In conclusion, our studies in human bronchial epithelial BEAS 2B cells showed that uncoated nanosized anatase TiO(2) and fine rutile TiO(2) are more efficient than SiO( 2)-coated nanosized rutile TiO(2) in inducing DNA damage, whereas only nanosized anatase is able to slightly induce micronuclei.

Preparation of nanoparticle dispersions for in-vitro toxicity testing.

Studies on potential toxicity of engineered nanoparticle (ENP) in biological systems require a proper and accurate particle characterization to ensure the reproducibility of the results and to understand biological effects of ENP. A full characterization of ENP should include various measurements such as particle size and size distribution, shape and morphology, crystallinity, composition, surface chemistry, and surface area of ENP. It is also important to characterize the state of ENP dispersions. In this study, four different ENPs, rutile and anatase titanium dioxides and short single- and multi-walled carbon nanotubes, were characterized in two dispersion media: bronchial epithelial growth medium, used for bronchial epithelial BEAS cells, and RPMI-1640 culture media with 10% of fetal calf serum (FCS) for human mesothelial (MeT-5A) cells. The purpose of this study was to determine the characteristics of ENPs and their dispersions as well as to compare dispersion additives suitable for toxicity tests and thus establish an appropriate way to prepare dispersions that performs well with the selected ENP. Dispersion additives studied in the media were bovine serum albumin (BSA) as a protein resource, dipalmitoyl phosphatidylcholine (DPPC) as a model lung surfactant, and combination of BSA and DPPC. Dispersions were characterized using optical microscopy and transmission electron microscopy. Our results showed that protein addition, BSA or FCS, in cell culture media generated small agglomerates of primary particles with narrow size variations and improved the stability of the dispersions and thus also the relevance of the in-vitro genotoxicity tests to be done.

In vitro sensitivity of normal human mesothelial and malignant mesothelioma cell lines to four new chemotherapeutic agents.

In this study, we used four human mesothelioma cell lines (M14K, M24K, M25K and M38K), one transformed human mesothelial cell line (MeT-5A) and one primary mesothelial culture (UPL) to test for in vitro sensitivity to docetaxel, paclitaxel, SN-38 [an active metabolite of irinotecan (CPT-11)] and gemcitabine, as single agents. Subconfluent cell cultures were treated with 2x10(-9), 5x10(-9), 10(-8), 2x10(-8) and 5x10(-8) M concentrations of each drug for 48 h. The sensitivity was measured in terms of cell viability using the Trypan blue exclusion method. All four drugs were potent inhibitors of mesothelioma cell growth, but cell lines from different patients diverged in their sensitivity to the individual agents. In most cases docetaxel, paclitaxel and SN-38 were more potent killers of mesothelioma cells than gemcitabine. The induction of DNA damage was investigated using the Comet assay; cells from two cell lines (M14K and M25K) were treated with subtoxic 10(-8) M concentrations of each drug for 4, 24 and 48 h. Each of the agents caused a slight increase in DNA single-strand breaks at a concentration of 10(-8) M.

Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection.

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.

Three-year follow-up of the use of a levonorgestrel-releasing intrauterine system in hormone replacement therapy.

BACKGROUND: The efficacy of a levonorgestrel-releasing intrauterine system in opposing endometrial proliferation and in preventing bleeding was studied in peri- and postmenopausal women receiving estrogen replacement therapy. METHODS: This was an open, non-controlled follow-up study of the use of a levonorgestrel-releasing intrauterine system during continuous estrogen replacement therapy carried out by using oral, transdermal or subdermal estradiol. The efficacy of the progestin therapy was evaluated by transvaginal ultrasonography and by examination of endometrial biopsy samples taken 20 months (mean, range 17 - 22; first evaluation) and 34 months (mean, range 31 - 38 months; second evaluation) after insertion of the levonorgestrel-releasing intrauterine system, and by studying patterns of bleeding. Twenty-five women participated in the first evaluation, and 29 in the second. RESULTS: Seventy-six percent of the women were amenorrheic at the first evaluation, and 79% at the second evaluation. Others had spotting for 1-2 days monthly or less often. The mean time until amenorrhea was reached was 6 months (range 2-13 months) after insertion of the levonorgestrel-releasing intrauterine system. The median endometrial thickness assessed by ultrasound was 2 mm at both evaluations. No signs of proliferation were observed in any of the endometrial samples. CONCLUSIONS: Local progestin delivery via a levonorgestrel-releasing intrauterine system was effective in suppressing the endometrium and in eliminating bleeding in women receiving estrogen replacement therapy, and the intrauterine progestin therapy was also well accepted.

Endometrial response to hormone replacement therapy as assessed by expression of insulin-like growth factor-binding protein-1 in the endometrium.

OBJECTIVE: To assess endometrial response to parenteral levonorgestrel in hormone replacement therapy by means of morphological criteria and immunohistochemical staining of insulin-like growth factor-binding protein-1 (IGFBP-1). DESIGN: Endometrial samples were collected from 35 postmenopausal women after 12 to 22 months of continuous combined estrogen-progestin therapy. All subjects were treated with parenteral progestin. A group of 8 women was treated with a subdermal levonorgestrel-releasing implant, and 27 women had a levonorgestrel-releasing intrauterine device (IUD). Sections of formalin-fixed paraffin-embedded biopsies were used for immunohistochemistry and after hematoxylin-eosin staining for routine histologic examination. RESULTS: Atrophic epithelium with pronounced decidual reaction in the stroma was detected by histologic examination in all endometrial samples obtained from 27 women treated with the levonorgestrel-releasing IUD. In contrast, the endometrium was proliferative in seven of eight (87.5 percent) biopsies obtained from women treated with the levonorgestrel-releasing implant. Immunoreactive IGFBP-1 was detected in decidualized stromal cells in all endometrial samples obtained during intrauterine levonorgestrel therapy, whereas only one of eight samples obtained from women treated with subdermal levonorgestrel exhibited weak staining for IGFBP-1. CONCLUSIONS: Our data show that both the morphological and biochemical response of post- menopausal endometrium to parenteral levonorgestrel was strikingly different, depending on the route of progestin administration, and that the decidual reaction and epithelial atrophy induced by intrauterine levonorgestrel were associated with expression IGFBP-1 in decidualized stromal cells.

Use of post-coital contraception in Finland is increasing.

PIP: In 1986, Finland registered a 4-pill regimen (50 mcg ethinyl estradiol and 250 mcg levonorgestrel in each pill) for use as a prescribed postcoital contraceptive. All regional primary health care centers provide contraceptive services free of charge, and the most popular form of postcoital contraception is this combined pill regimen. While data are not collected on the numbers of women who seek postcoital contraception from the clinics, prescription sales of the package have increased (to 25,000 in 1994), and the number of abortions has decreased, especially among girls 15-19 years old. In addition to making postcoital contraception available, efforts have been underway to educate the public, especially young people, about the method and to train health personnel in its use. The remaining obstacles to the promotion of postcoital contraception are claims that the procedure reduces use of regular contraception and makes it harder for young women to refuse to engage in sexual intercourse. The pill packaging also indicates that treatment should begin within 48 hours of unprotected intercourse. This should be changed to a time limit of 72 hours. Condom packaging should also point to the availability of postcoital contraception if the condoms fail. The public also needs to be informed that use of postcoital contraception does not constitute a drug-induced abortion. Since there are no absolute contraindications or serious side effects associated with the use of the regimen, postcoital contraception should be available without prescription.^ieng

Intrauterine and subdermal progestin administration in postmenopausal hormone replacement therapy.

OBJECTIVE: To compare the effects of intrauterine and subdermal administration of levonorgestrel on control of bleeding and on the endometrium in postmenopausal hormone replacement therapy. INTERVENTIONS: Nineteen women started continuous oral E2 valerate therapy (2 mg daily) together with continuous parenteral progestin therapy. The subjects randomly received either a subdermal levonorgestrel-releasing implant (n = 9) or an intrauterine device (IUD) releasing levonorgestrel (n = 10). MAIN OUTCOME MEASURES: Serum concentrations of estrone, E2, FSH, sex hormone-binding globulin (SHBG) and levonorgestrel were followed. Endometrial biopsies and transvaginal ultrasonography were used to evaluate the endometrium. The subjects kept daily records of bleeding. The observation time was 1 year. RESULTS: Serum concentrations of the hormones mentioned above and SHBG were similar in both groups during the observation time, but the patterns of bleeding differed. In the IUD group there were 0.9 days (mean, range 0 to 4 days) of spotting and no days of bleeding during the last month of the follow-up year. In the implant group there were 8 days (mean, range 0 to 25 days) of spotting and 3.4 days (mean, range 0 to 14 days) of bleeding. In histological examination there was uniform atrophy in the endometrial samples from the IUD group, and a weak or absent progestin effect in the implant group. CONCLUSIONS: In spite of similar serum levonorgestrel concentrations, local intrauterine administration of levonorgestrel resulted in better control of bleeding and in more effective endometrial suppression than subdermal administration.

Sustained-release estradiol implants and a levonorgestrel-releasing intrauterine device in hormone replacement therapy.

OBJECTIVE: Efficacy and acceptability of continuous combined parenteral hormone replacement consisting of subdermal estradiol delivery and intrauterine progestin delivery was studied. STUDY DESIGN: Thirty-six volunteer postmenopausal women seeking treatment for climacteric symptoms participated in this open, randomized study. The subjects received either one or three subdermal implants with a controlled daily release of estradiol in vitro. Progestin therapy was carried out with a levonorgestrel-releasing intrauterine device. Climacteric symptoms and serum concentrations of estrone, estradiol, follicle-stimulating hormone, and sex hormone-binding globulin were followed up for 1 year. The subjects kept daily records of bleeding. RESULTS: Serum estradiol concentrations with the set of three implants were stable during the follow-up period; the range of mean serum estradiol concentrations was 45 to 57 pg/ml. Four women, all from the group with one implant, discontinued the study after 6 months because of the return of climacteric symptoms. In spite of different daily estradiol doses, the patterns of bleeding were similar in both groups. At the end of the follow-up year 72% of the women had had no bleeding or spotting in the previous 3 months. CONCLUSION: Our results suggest that by combining subdermal and intrauterine steroid delivery systems postmenopausal hormone replacement therapy can be carried out successfully without daily effort and with minimal or no bleeding.

Sustained-release subdermal estradiol implants: a new alternative in estrogen replacement therapy.

OBJECTIVE: This trial was a therapeutic and pharmacokinetic dose-finding study with subdermal implants that showed a constant in vitro daily release rate of estradiol. The aim was to find the daily release rate of estradiol that produces serum estradiol concentrations comparable with those obtained with transdermal estradiol at 0.05 mg/day. STUDY DESIGN: The study was an open crossover comparison of transdermal and subcutaneous delivery systems. Thirty-six postmenopausal (serum follicle-stimulating hormone concentration > 30 IU/L before commencing the study) were treated with transdermal estradiol for 4 weeks. Immediately after this transdermal estradiol delivery was replaced by either one or three estradiol implants. The serum concentrations of estrone, estradiol, follicle-stimulating hormone, and sex hormone-binding globulin were followed up for 8 weeks on implant therapy and compared with those during transdermal estrogen administration. To oppose the estrogen effect on the endometrium, each patient received an intrauterine device releasing a progestin, levonorgestrel. RESULTS: The serum estradiol concentrations achieved with three implants were comparable with those during transdermal administration of estradiol at 0.05 mg/day. Suppression of follicle-stimulating hormone was also better maintained with three implants compared with one. CONCLUSIONS: This study shows that constant release profiles can be achieved with nonbiodegradable estradiol implants. The results suggest that a single application should give sufficient estrogen substitution for more than a year.

Postmenopausal oestrogen replacement therapy with subcutaneous oestradiol implants.

Ten postmenopausal patients were treated by means of subcutaneous oestradiol-releasing silastic implants. Half of the patients received 3 implants, each containing 12 mg oestradiol valerate (E2V), while the other half received 4 implants, each containing 27 mg oestradiol benzoate (E2B). Progestogen was added to the treatment for 14 days, 6 weeks after implant insertion and every fourth week thereafter. Serum levels of oestrone (E1), oestradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were followed up. The effects on endometrial thickness, uterine volume and breast tissue were evaluated by ultrasound, mammography also being used for breast examination. The follow-up period was 24 weeks, but the implants were not removed until the climacteric symptoms reappeared. E1 and E2 levels remained higher and gonadotrophin levels lower than the pretreatment values during the 24-week follow-up period. Oestrogen effects were seen in both the uterus and the breasts. Both types of implant were effective in relieving climacteric symptoms. The mean time for symptom return was 10 months (range 6-18 months) in the E2V group and 8 months (range 4-12 months) in the E2B group. Our results indicate that nonbiodegradable controlled-release oestrogen implants offer a safe, effective, convenient and well-accepted alternative means of administering oestrogen replacement therapy.

Endometrial effect of transdermal estradiol and progestin ST-1435 in postmenopausal women.

OBJECTIVE: To study the endometrial effect of the transdermal synthetic progestin ST-1435. DESIGN: Prospective. SETTING: City Maternity Hospital, Helsinki, Finland. PATIENTS: Eleven postmenopausal women used transdermal estradiol (E2) patches for 6 weeks immediately before a vaginal operation for prolapse. For the last 10 days, 1 mg of ST-1435 transdermally in a gel was combined to the treatment. MAIN OUTCOME MEASURES: Blood samples were taken to follow serum concentrations of E2, follicle-stimulating hormone, and ST-1435. Endometrial samples for histologic examination were collected during the operation to evaluate the effect of the progestin. RESULTS: Transdermal absorption of ST-1435 resulted in reasonably constant serum concentrations of ST-1435 in each subject. A progestin effect on the endometrium was seen in 9 of 10 samples obtained. One sample did not show any progestin effect in spite of adequate ST-1435 levels, but this patient's E2 concentrations were low. CONCLUSIONS: When the estrogen stimulation was adequate, the transdermal ST-1435 induced a progestin effect on the endometrium, i.e., it had an end-organ effect.