RESUMEN
Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.
Asunto(s)
Mutágenos/toxicidad , Quinacrina/toxicidad , Animales , Células CHO , Aberraciones Cromosómicas , Cricetinae , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Esterilización ReproductivaRESUMEN
The rat is the preferred species for reproductive toxicity testing. The inclusion of measures of rat sperm quality, such as motility and morphology, into reproductive test protocols often increases the sensitivity of the test to detect effects, and provides the toxicologist and risk assessor with valuable information about the nature of the reproductive toxicity of the test substance. Technical advances in computer-aided sperm analysis have made it possible to evaluate motion characteristics of rat spermatozoa. This technology can provide an objective means of classifying the motion of rat spermatozoa as progressive or non-progressive, as required in test protocols. More specific tests of rat sperm function are being applied for the purpose of evaluating modes and mechanisms of toxicant action. Computer-aided sperm analysis can be used to evaluate sperm motion during cultures that support sperm capacitation and to identify hyperactivated spermatozoa. Under the same culture conditions, acrosome-specific stains can be used to identify effects of toxicants on the acrosome reaction. These approaches, in combination with in vitro fertilization in rats, can pinpoint sperm functional deficits and thereby assist the toxicologist in addressing hypotheses regarding the cellular-molecular bases of toxicant-induced male infertility.
Asunto(s)
Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Pruebas de Toxicidad/métodos , Reacción Acrosómica , Animales , Evaluación Preclínica de Medicamentos , Femenino , Fertilización In Vitro , Guías como Asunto , Humanos , Inseminación Artificial , Masculino , Ratas , Capacitación Espermática , Motilidad Espermática , Testículo/citología , Testículo/efectos de los fármacos , Testículo/fisiología , Estados Unidos , United States Environmental Protection Agency , United States Food and Drug AdministrationRESUMEN
The aim of this study was to use computer-assisted sperm analysis (CASA) to examine changes in motion parameters of rat spermatozoa incubated under culture conditions that support IVF. Rat cauda epididymal spermatozoa were evaluated in six replicate experiments, at 0 and 4h of incubation. CASA was conducted at 60 Hz on digital 1s tracks ( approximately 100 spermatozoa/rat). Mean values of CASA parameters that describe the vigour of spermatozoa [curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] increased, while those indicating progressiveness [straight line velocity (VSL), linearity (LIN) and straightness (STR)] decreased between 0 and 4 h. Visual inspection of sperm tracks after 4 h of incubation revealed classical hyperactivation patterns. Bivariate models were evaluated to objectively define the subpopulation of hyperactivated (HA) spermatozoa. Of all models considered, ALH and LIN, VCL and LIN, BCF and LIN, VCL and BCF, and VCL and ALH showed significant changes in the percentage of HA spermatozoa after the 4 h incubation period. The efficacy of detecting HA spermatozoa was evaluated using sperm tracks that were visually classified as HA or progressive. VCL and LIN provided the most accurate prediction of HA spermatozoa. It was concluded that analysis of CASA data using bivariate models could be used to detect and monitor hyperactivation in rat spermatozoa.
Asunto(s)
Procesamiento Automatizado de Datos , Motilidad Espermática , Animales , Células Cultivadas , Fertilización In Vitro/métodos , Masculino , Modelos Biológicos , Ratas , Ratas Sprague-DawleyRESUMEN
Previously we reported that the 55-kDa fertility-associated protein in Holstein bull seminal plasma (SP) is osteopontin (OPN). The objective of the present study was to localize OPN in tissues and fluids in the Holstein bull reproductive tract to determine its origin. Antisera generated against human recombinant OPN, as well as antiserum prepared against purified bovine seminal plasma OPN, reacted with protein in SP, accessory sex gland fluid, seminal vesicle fluid, ampullary fluid, and urine using one- and two-dimensional SDS-PAGE Western blot analysis. However, these antisera failed to detect OPN in cauda epididymal fluid or solubilized sperm membranes. Immunofluorescence histochemistry localized OPN in the lumen and epithelial cells of the seminal vesicle and ampulla, but not in tissues of testis, epididymis, prostate, and bulbourethral gland. OPN was not detected immunohistochemically in epididymal, ampullary, or ejaculated sperm treated with or without Triton X-100. We concluded that the primary sources of OPN in bull SP are the seminal vesicles and ampulla.
Asunto(s)
Líquidos Corporales/química , Bovinos , Genitales Masculinos/química , Sialoglicoproteínas/análisis , Animales , Western Blotting , Membrana Celular/química , Electroforesis en Gel de Poliacrilamida , Epidídimo/química , Técnica del Anticuerpo Fluorescente , Genitales Masculinos/metabolismo , Humanos , Masculino , Osteopontina , Proteínas Recombinantes/inmunología , Semen/química , Vesículas Seminales/química , Sialoglicoproteínas/orina , Espermatozoides/ultraestructuraRESUMEN
Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.
Asunto(s)
Fertilidad , Semen/química , Sialoglicoproteínas/análisis , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Densitometría/métodos , Electroforesis en Gel Bidimensional , Glicosilación , Masculino , Datos de Secuencia Molecular , Osteopontina , Fosforilación , Análisis de Secuencia , Sialoglicoproteínas/químicaRESUMEN
Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. In this work, we investigated the roles of veratryl alcohol and lignin in the ligninolytic system of P. chrysosporium BKM-F-1767 cultures grown under nitrogen-limited conditions. Cultures supplemented with 0.4 to 2 mM veratryl alcohol showed increased lignin peroxidase activity. Addition of veratryl alcohol had no effect on Mn-dependent peroxidase activity and inhibited glyoxal oxidase activity. Azure-casein analysis of acidic proteases in the extracellular fluid showed that protease activity decreased during the early stages of secondary metabolism while lignin peroxidase activity was at its peak, suggesting that proteolysis was not involved in the regulation of lignin peroxidase activity during early secondary metabolism. In cultures supplemented with lignin or veratryl alcohol, no induction of mRNA coding for lignin peroxidase H2 or H8 was observed. Veratryl alcohol protected lignin peroxidase isozymes H2 and H8 from inactivation by H2O2. We conclude that veratryl alcohol acts as a stabilizer of lignin peroxidase activity and not as an inducer of lignin peroxidase synthesis.