Ana Rita da Silva

Contraception with long acting subdermal implants: I. An effective and acceptable modality in international clinical trials

This paper presents results of a double blind, multi-centered and multi-national study of two progestin only subdermal implants used for contraception. A regimen of six capsules of levonorgestrel (Ng) used by 492 women had a net cumulative 12-month pregnancy rate of 0.6 percent and a continuation rate of 74.6 percent. 498 women used six capsules of norgestrienone (R2010) and experienced a net cumulative 12-month pregnancy rate of 3.5 percent and a continuation rate of 79.4 percent. The difference in the pregnancy rate was significant at P less than 0.01, while there was no significant difference in the continuation rates. Menstrual problems were the principal reason for termination of the levonorgestrel regimen, accounting for approximately half of all terminations. There were significantly fewer menstrual problems among users of the norgestrienone (R2010) capsules; the net cumulative 12-month termination rate for this reason was 4.3 percent. Results are compared with continuation and termination rates for acceptors of the Copper T 200 at the same clinics. The low pregnancy rate and reasonably high continuation rate of the norgestrel implants coupled with the fact that the expected effective lifetime of a set of capsules is of the order of 3-5 years appears to warrant further development of this contraceptive regimen.

One-Year Contraception with Norgestrienone Subdermal Silastic Implants *

Silastic capsules of Norgestrienone 17 alpha-ethinyl-17-hydroxy-estra-4 9 11-trien-3-one were inserted subcutaneously in the anterior forearm or in the upper gluteal region of 503 women of childbearing age. Approximately 30% of the women had not received any contraceptive steroids previously whereas 70% had been on contraceptive pills for 1 month to 8 years. The women were divided into 4 groups: Group 1 received 1 capsule and was followed for 6 months; Group 2 received 2 capsules and was observed for 10 months; Group 3 received 3 capsules and was observed for 12 months; and Group 4 received 4 capsules and was observed for 13 months. Each capsule contained 47 mg of the compound. In Groups 1 and 2 the pregnancy rate was high and the contraceptive effect lasted only a short time. In Groups 3 and 4 contraceptive effectiveness was high and lasted a minimum of 10 and 12 months respectively. In Group 4 which was the largest 249 women were observed for 1570 cycles. Only 1 pregnancy occurred in this group. Breakthrough bleeding occurred in less than 5% of all cycles and amenorrhea was reported by 30% of the patients in less than 10% of all cycles. No pain or infection at the site of implantation was reported.

Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells

Plants of Crotalaria genus (Leguminosae) present large amounts of the pyrrolizidine alkaloid monocrotaline (MCT) and cause intoxication to animals and humans. Therefore, we investigated the MCT-induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model. The comet test showed that 24h exposure to 1-500microM MCT and 500microM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42-64% and 53%, respectively. Cells exposed to 100-500microM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1-10microM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1-10microM). Apoptosis was also evidenced in cells treated with 100-500microM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500microM MCT. The data obtained provide support for heterogenic and multipotential effects of MCT on GL-15 cells, either interfering on cell growth and cytoskeletal protein expression, or inducing DNA damage and apoptosis and suggest that the response of glial cells to this alkaloid might be related to the neurological signs observed after Crotalaria intoxication.

Ovulation inhibition following vaginal administration of pills containing norethindrone and mestranol

Plasma levels of estradiol and progesterone were investigated in women using daily vaginal pills containing 1 mg norethindrone and 50 mcg mestranol. Of 13 treatment cycles in ten women using one vaginal pill daily, six were ovulatory and seven anovulatory. All 12 cycles in ten women using two vaginal pills daily were anovulatory.

Cytotoxicity of catechol towards human glioblastoma cells via superoxide and reactive quinones generation

It is known that the exposure to benzene in the petroleum industry causes lympho-haematopoietic cancer among workers. However, there is little data concerning the toxicity of benzene to the central nervous system. Benzene easily penetrates the brain where it is metabolized to catechol. Since catechol autoxidizes in physiological phosphate buffer, we hypothesized that it could be toxic towards glial cells due to the generation of reactive oxygen species and quinones. In this work we studied the cytotoxic properties of catechol towards human glioblastoma cells. We found that catechol was toxic towards these cells after 72 hours and this toxicity was related to the formation of quinones. Catechol at 230µM killed 50% of cells. The catechol-induced cytotoxicity was prevented by the addition of 100U superoxide dismutase, which also inhibited the formation of quinones. These data suggest that catechol induces cytotoxicity via the extracellular generation of superoxide and quinones.

Comparative study on the efficacy and acceptability of two contraceptive pills administered by the vaginal route : an international multicenter clinical trial

The efficacy and acceptability of two widely used oral contraceptive tablets, one containing 250 mg levonorgestrel and 50 µg ethinyl estradiol and the other containing 150 μg desogestrel and 30 µg ethinyl estradiol, administered by the vaginal route were compared in 1055 women studied over 12,630 woman‐months of vaginal contraceptive pill use. This multicenter clinical trial was performed in nine countries of the developing world by the “South to South Cooperation in Reproductive Health,” an organization founded by scientists from the Third World working in the area of reproductive health, and the study was developed and coordinated by one of these centers. The findings of this study confirm the efficacy of both these tablets when administered by the vaginal route. Involuntary pregnancy rates at 1 year of 2.78 for subjects in the levonorgestrel group and 4.54 for subjects the desogestrel group showed no statistically significant difference between the two groups. However, total discontinuation rates of 47.01 for subjects in the levonorgestrel group and 56.33 for subjects in the desogestrel group showed a statistically significant difference between the two groups, and discontinuation rates attributable to prolonged bleeding of 0.6 for subjects in the levonorgestrel group and 3.2 for subjects in the desogestrel group were also significantly higher in the group of subjects using the desogestrel vaginal contraceptive pill. Blood pressure remained at admission values throughout treatment. A statistically significant weight increase from admission values occurred in both groups of subjects.

Assessment of neurotoxicity of monocrotaline, an alkaloid extracted from Crotalaria retusa in astrocyte/neuron co-culture system

Studies have shown cases of poisoning with plants from the genus Crotalaria (Leguminosae) mainly in animals. They induce damages in the central nervous system (CNS), which has been attributed to toxic effects of the pyrrolizidine alkaloid (PA) monocrotaline (MCT). Previously we demonstrated that both MCT and dehydromonocrotaline (DHMC), its main active metabolite, induce changes in the levels and patterns of expression of the main protein from astrocyte cytoskeleton, glial fibrillary acidic protein (GFAP). In this study we investigated the effect of MCT on rat cortical astrocyte/neuron primary co-cultures. Primary cultures were exposed to 10 or 100 μM MCT. The MTT test and the measurement of LDH activity on the culture medium revealed that after 24h exposure MCT was not cytotoxic to neuron/astrocyte cells. However, the cell viability after 72 h treatment decreased in 10-20%, and the LDH levels in the culture medium increased at a rate of 12% and 23%, in cultures exposed to 10 or 100 μM MCT. Rosenfeld staining showed vacuolization and increase in cell body in astrocytes after MCT exposure. Immunocytochemistry and Western blot analyses revealed changes on pattern of GFAP and βIII-tubulin expression and steady state levels after MCT treatment, with a dose and time dependent intense down regulation and depolarization of neuronal βIII-tubulin. Moreover, treatment with 100 μM MCT for 12h induced GSH depletion, which was not seen when cytochrome P450 enzyme system was inhibited indicating that it is involved in MCT induced cytotoxicity in CNS cells.

Clastogenic effect of extracts obtained from Crotalaria retusa L. and Crotalaria mucronata Desv. on mouse bone marrow cells

This work has evaluated the clastogenicity of six extracts (tea and aqueous extract of leaves, tea, aqueous and methanolic extracts of dried fruit, and tea of unripe fruit) obtained from Crotalaria retusa L. and three extracts (tea and methanolic extract of dried fruit, and tea of unripe fruit) obtained from Crotalaria mucronata Desv. The extracts were injected intraperitoneally into mice, and the animals were killed 24 h after treatment for preparation of bone marrow cells. The extracts obtained from fruits of Crotalaria retusa were found to cause a dose-dependent increase in the frequency of chromosomal aberrations in mice. On the other hand, no statistically significant increase in the frequency of aberrant cells was observed for the animals treated with leaf extracts obtained from Crotalaria retusa and with extracts from fruits of Crotalaria mucronata. The possibility that the pyrrolizidine alkaloid, monocrotaline, present in Crotalaria retusa exerts a clastogenic effect on mouse bone marrow cells is discussed. Our conclusion is based on studies using intraperitoneal treatments. Effects of oral exposure to extracts of Crotalaria retusa are unknown.

Conception control by vaginal administration of pills containing ethinyl estradiol and dl-norgestrel

One hundred twenty-four women of reproductive age have used vaginal pills containing 50 micrograms dl-norgestrel and 35 micrograms ethinyl estradiol to prevent conception for periods ranging from 6 to 20 months. One thousand four hundred thirty-eight woman-months were recorded. No pregnancies occurred. Cycle control was good. Bleeding usually lasted 3 to 5 days, and the interval between withdrawal bleeding events was 26 to 30 days in 86% of the cycles. Amenorrhea, breakthrough bleeding, and spotting occurred rarely. The continuation rate at 1 year was 64%.

Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures.

Dehydromonocrotaline (DHMC) is the main monocrotaline active cytochrome P450s metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 microM DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 microM, and induced membrane damages at 500 microM. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 microM DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 microM), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/-30%) of the astrocytes exposed to 100-500 microM DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependent effect of DHMC on cultures of rat cortical astrocytes, leading to apoptotic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria.