Nieves Martin-Alguacil

Comparisons of two in vitro cytotoxicity assays—The neutral red (NR) and tetrazolium MTT tests

The neutral red (NR) and tetrazolium MTT in vitro cytotoxicity assays were compared for 28 test agents of widely varying potency using the BALB/c mouse 3T3 fibroblast cell line as the bioindicator. For any given cell density in the microtitre plate well, the optical density absorbance with the NR assay was about twice that obtained with the MTT assay. However, there was good agreement (r = 0.939) between the ranking of the test agents on the basis of midpoint cytotoxicity values (NR(50) and MTT(50)), although the assays were based on different physiological endpoints. Nevertheless, the two assays were found to differ in sensitivity for a few test agents, such as chloroquine sulphate and several antineoplastic drugs. Both assays were capable of demonstrating a reduction in the cytotoxicity of methotrexate by leucovorin.

Rapid chemosensitivity assay with human normal and tumor cells in vitro

SummaryNeutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development.

Success of Treatment Modalities for Labial Fusion: A Retrospective Evaluation of Topical and Surgical Treatments

STUDY OBJECTIVE Standard treatment for girls with labial fusion has included topical estrogen cream, manual separation, or surgery. Side effects may limit the use of topical estrogen. Betamethasone has recently shown efficacy at separating labial fusion. Local irritation and inflammation may be an initiator of labial fusion. No adverse effects of betamethasone treatment have been documented. Long-term side effects are unknown. This study compares therapies for conservative management of labial fusion for efficacy and focuses on the response rate, time to separation, recurrence, and side effects of treatment. DESIGN A retrospective chart review. PARTICIPANTS One hundred fifty-one prepubertal girls, mean age 3 years (range 0.25-8.75 years) diagnosed with labial fusion. MAIN OUTCOME MEASURES To investigate the incidence of related symptoms, length of topical estrogen or betamethasone treatment, side effects, rate of successful separation, rates of recurrence, percentage requiring surgery, and postoperative outcomes in patients with labial adhesion who underwent treatment. RESULTS Of 151 patients with labial adhesion, 11 (7.3%) presented with urinary frequency, 30 (19.9%) with urinary tract infections, 13 (8.6%) with vaginitis, and 19 (12.6%) with post-void dripping. When compared to patients treated with betamethasone (1.3 months), patients treated primarily with premarin took nearly twice as long (2.2 months) for resolution of their adhesions. Rates of recurrence were lower for patients receiving betamethasone therapy. Side effects for estrogen therapy included breast budding and vaginal bleeding, and for betamethasone, local irritation was reported. Some patients went on to surgery and experienced recurrence after surgery. CONCLUSION Initial comparison of topical estrogen and betamethasone treatment of labial fusion suggests that betamethasone may separate fusion quicker with less recurrence and fewer side effects than topical estrogen therapy.

Clitoral sexual arousal: neuronal tracing study from the clitoris through the spinal tracts.

PURPOSE Although genital tactile stimulation is regarded as a precursor to sexual arousal and a recognized initiator of central nervous system arousal, specific afferent neural pathways transmit sensory stimuli of arousal, beginning at the epithelial level on the clitoris and following the course of arousal stimuli through the central nervous system. Limited knowledge exists of the pathway from the cutaneous receptors of nerves originating in the epithelial tissue of the clitoris and continuing to spinal cord afferents. Such information may contribute to an understanding of sexual arousal, particularly in female vertebrates. We further defined the neural pathways and mechanisms responsible for arousal originating in the epithelium of the clitoris as well as related neural pathways to the spinal cord in a murine model. MATERIALS AND METHODS We performed a comprehensive review of the published relevant clinical and histological material from human and nonhuman vertebrate studies. In 29 adult female C57B1/6 mice the distribution of pelvic nerves and vessels was mapped. Gross dissection of 4 female mice was facilitated by resin injection of the vascular system in 2. Neuronal tracing was performed in 25 mice that received clitoral injection of wheat germ agglutinin-horseradish peroxidase into the clitoris and were sacrificed after 72 to 96 hours. The spinal cord and periclitoral tissue were removed and fixed. Immunohistochemistry was performed. RESULTS Gross anatomy of the mouse clitoris showed that pudendal and hypogastric nerves have a major role in the innervation of the external genitalia. Neuronal tracing revealed that the greatest nerve density was noted in the L5/6 spinal cord. The distribution extended from S1 to L2 with no labeling seen in the L3 spinal cord. Wheat germ agglutinin-horseradish peroxidase labeling was seen caudal in levels S1 through L4 and rostral in L2. CONCLUSIONS Understanding the neuroanatomy of the clitoris using a murine model may provide a valuable tool for the study of sexual arousal disorders and the further understanding of sexual function related to neural pathologies and trauma.

Clitoral sexual arousal: an immunocytochemical and innervation study of the clitoris

To further define neural pathways and mechanisms responsible for the arousing properties of the epithelium of the clitoris as well as related neural pathways associated with sexual arousal in a murine model.

Arsenic-selenium interactions determined with cultured fish cells.

A fibroblastic cell line derived from gill tissue (designated BG/G) and an epithelioid cell line derived from fin tissue (designed BG/F) of bluegill sunfish (Lepomis macrochirus) were used as the bioindicators in toxicity experiments. The neutral red in vitro cytotoxicity assay served as the endpoint. In both cell lines the sequence of observed cytotoxicity was arsenite greater than arsenate greater than selenite greater than selenate, with each cell type exhibiting comparable ranking of midpoint toxicity (NR50) concentrations. Antagonistic interactions were noted between combinations of arsenics and seleniums. Thus, selenate and selenite (at nontoxic levels) reduced, but did not eliminate, the acute cytotoxicities of arsenate and, to a lesser extent, of arsenite.

Innervation of the Labia Minora of Prepubertal Girls

INTRODUCTION Surgical and histologic sources of information give little reference to innervation, vascular, and epithelial details of the labia minora. Little is known about areas of nerve density, epithelial qualities, and vascular compartments of the labial minora that contribute to sexual arousal and orgasm. Surgical procedure development and counsel about surgical risks related to labioplasty and surgical flaps created from labial tissue may be based on inadequate information. METHODS Labial samples from 10 normal girls (aged 2-9 years) who underwent surgery for labial fusion utilized waste tissue strips for immunohistochemical identification of S-100 and neuronal nitric oxide synthase (nNOS) in the labia minora. RESULTS Vascular and lymphatic plexus lie within the reticular dermis, which contains a dense mesh of nerve fibers with a higher concentration of nerve fiber at the level of the subepithelial plexus. Dense innervations are located at the epidermis, extending along the basal and spinous layers of the epithelium of labia minora. Nerve bundles in the papillary dermis are associated with sebaceous and eccrine glands and nerve terminals located throughout the epithelium. The introital epithelium of the labia minora is highly innervated with widespread and intense staining, detected in the introital border of the labia minora versus the external one. The dermis appeared to display S-100 and nNOS immunolabelling. S-100 was also immunopositive in the epidermis. CONCLUSION Labia minora is highly innervated along its entire edge. Related vascular compartment tissue involved in engorgement during sexual arousal makes this tissue important for sexual response. Labioplasty risks removal of tissue with an important contribution to sensory sexual arousal. Movement of labial tissue during genitoplasty may have different sensory outcomes dependent on which labial surface is used.

Genetic Mechanisms in Neural and Hormonal Controls over Female Reproductive Behaviors

Building on well-established mechanisms that produce the primary female mating behavior, lordosis, research is extending into mechanisms for sexual arousal. Genes and neurochemcal pathways supporting sexual arousal are reviewed, and four neurochemical/biophysical routes by which generalized arousal could influence sexual arousal are charted.

Oestrogen receptors and their relation to neural receptive tissue of the labia minora

Associate Editor

In vitro response of the brown bullhead catfish cell line, BB, to aquatic pollutants

Established cell lines from brown bullhead catfish (BB) and rainbow trout (RTG-2) and primary cultures of cells derived from gill, fin, and gonad tissues from brown bullhead catfish were evaluated for use as bioindicators in the neutral red cytotoxicity assay. The BB and RTG-2 cells were compared after a 1 day exposure to chlorinated pesticides and after a 6-day exposure to various polycyclic aromatic hydrocarbons. The BB cells were more sensitive to both classes of chemicals. The sequence of toxicity for the BB cells was 4,4′-DDD, 4,4′-DDT > aldrin, 4,4′-DDE and 7,12-dimethylbenz(a)anthracene (DMBA) > 3-hydroxybenzo(a)pyrene (3-OH-B(a)P) > benzo(a)pyrene (B(a)P). For the RTG-2 cells, the sequence was aldrin > 4,4′-DDD > 4,4′-DDT > 4,4′-DDE and 3-OH-B(a)P > DMBA > B(a)P. The BB cells were also sensitive to benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)anthracene, and trans-7,8-di-hydro-7,8-dihydroxybenzo(a)pyrene. The responses of BB and RTG-2 cells were compared with those of primary cultures after a 1 day exposure to B(a)P. After 1 day of exposure, the RTG-2 cells and primary cultures were more sensitive than the BB cells to B(a)P. Apparently, after 1 day of incubation the RTG-2 and primary cells metabolized greater amounts of B(a)P to cytotoxic metabolites, than did the BB cells. However, by 6 days of incubation, the BB cells were more sensitive to B(a)P than were the RTG-2 cells. A 6-day exposure to B(a)P was not performed with the primary cell cultures.