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R Bechter

Publications and source records attributed to R Bechter.

At least 19 recordsLinked to original sources
PubMed1998

Flow cytometry as a sensitive tool to assess testicular damage in rat.

The aim of this study was to compare results obtained by flow cytometry (FCM) with those obtained from testicular histopathology with regard to testicular damage following acute exposure of adult rats to the known testicular toxicant, methoxyacetic acid (MAA). Special emphasis was given to defining the sensitivity of three-parameter FCM compared with testicular histopathology. Furthermore, the effect on the male reproductive system of a single oral dose of MAA was evaluated with traditional methods, e.g. testicular sperm head counts, and organ weights. Adult, male Han/Wistar rats were randomly assigned to four groups of ten animals to be treated with a single dose of 0, 65, 325 and 650 mg MAA/kg body wt. (p.o., gavage). The animals were killed 2 days after treatment, and testicular and epididymal weights were recorded. One testis and the corresponding epididymis were used for histopathology. The other testis was used partly to determine sonication-resistant, testicular sperm-head counts (SHC), and partly for enzymatic digestion followed by FCM. The results obtained in this study are in agreement with the literature, and show that, in the adult male rat, 2 days after administering a single oral dose of MAA, specific depletion of spermatocytes is evident. Detectable testicular effects were produced by the high (650 mg/kg body wt.) and mid (325 mg/kg body wt.) doses, whilst the low dose (65 mg/kg body wt.) did not produce any noticeable effect. There was a strong correlation between results obtained by FCM and those obtained by testicular histopathology, and no difference in sensitivity between the two methods was observed. In summary, three-parameter FCM represents a sensitive and reliable method for the detection of testicular injury in the rat. It requires only small amounts of tissue, and the sensitivity was shown to be similar to that of histopathology. Moreover, FCM has the advantages of being quick and objective, which permits large numbers of cells to be analysed. The potential use of this method as a fast screening tool for testicular toxicity in routine toxicology studies should be considered.

Acetates↗
PubMed1997

Three-parameter flow cytometric analysis of rat spermatogenesis.

Mammalian spermatogenesis is a complex process which is not yet fully understood. In this paper we describe the analysis of rat spermatogenesis by means of 3-parameter flow cytometry. Since the analysis of DNA content only provides sufficient information for the identification of 4 cell populations, additional parameters were combined with propidium iodide (PI) staining. Immunostaining of the intermediate filament vimentin allowed the identification of somatic (vimentin positive) and germ (vimentin negative) cells. Utilizing the combination of DNA and vimentin staining, we have been able to quantitate the somatic cells present in a testicular cell suspension and to analyze somatic and germinal cells separately. Furthermore, the addition of mitochondrial staining with the fluorochrome nonyl acridine orange (NAO) allowed several cell subpopulations within each ploidy group to be distinguished. After 3-color staining and subsequent cell sorting, 11 testicular cell subpopulations could be identified: somatic cells, and 10 subtypes of germinal cells. The method described in this paper represents a valuable tool for the evaluation of spermatogenesis in both normal and perturbed situations.

Acridine Orange↗
PubMed1993

In vitro embryotoxicity of N-methyl-N-(7-propoxynaphthalene-2-ethyl)hydroxylamine (QAB): evidence for N-dehydroxylated metabolite as a proximate dysmorphogen.

The rat conceptus biotransforms N-methyl-N-(7-propoxynaphthalene-2-ethyl)hydroxylamine (QAB) in vitro to 7-propoxynaphthalen-2-ylacetic acid (QAA) and six more (M1 to M6) metabolites. Thus far, M4 has been identified as N-demethyl-QAB and M6 as N-desoxy-QAB. We investigated which of these two metabolites might be involved in QAB-embryotoxicity in vitro. Conceptuses were cultured from day 9.5 to 11.5 post-coitum, and were exposed to N-demethyl-QAB or N-desoxy-QAB either in the culture medium or by microinjection directly into the amniotic cavity. When added to the culture medium, N-demethyl-QAB (No Observed Adverse Effect Level, NOAEL, for growth 122 microM and for differentiation 41 microM) was less active than QAB itself (NOAEL for growth and differentiation 12 microM). N-desoxy-QAB caused severe growth retardation and an impairment of differentiation at a concentration of 11 microM (NOAEL 3.6 microM). As regards causing anomalies, the NOAEL of N-demethyl-QAB (41 microM) was 10-fold higher than that of QAB (NOAEL 3.9 microM) and that of N-desoxy-QAB (NOAEL 3.6 microM). At an intraamniotic concentration of 0.7 mM, N-demethyl-QAB caused no effects on growth and differentiation and no increase of anomalies was observed, whereas QAB and N-desoxy-QAB each elicited an increase in dysmorphogenic embryos at equimolar concentrations without affecting growth and differentiation. It is, therefore, concluded that N-desoxy-QAB, but not N-demethyl-QAB, could be a proximate dysmorphogen responsible for the embryotoxicity/teratogenicity of QAB in vitro.

Abnormalities, Drug-Induced↗
PubMed1992

Microinjections of cultured rat conceptuses: studies with 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide.

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.

Amnion↗
PubMed1992

Teratogenicity of arotinoids (retinoids) in the rat whole embryo culture.

Structural modifications of the arotinoid molecule RO 13-7410 led to a difference in the teratogenic potencies of more than five orders of magnitude in mice in vivo and in micromass cultures of rat embryonic limb bud cells (Kistler et al. 1990). Five of these retinoids were selected and tested in rat whole embryo culture to determine the suitability of this in vitro test system for the identification of potentially non-teratogenic derivatives among this class of chemicals. The highest concentrations of the compounds with no effects (NOAEL) on general conceptus growth, on differentiation and on the frequency of dysmorphogenic embryos in vitro were compared with the lowest effective teratogenic doses in vivo (LOAEL) or with the concentrations leading to 50% inhibition of limb bud cell differentiation (IC50) in vitro. NOAEL's for the parameters of conceptus development ranged from 10(-5) micrograms/ml (0.03 nM) to 10 micrograms/ml (28.7 microM) for the compounds tested. These correlated very well with LOAEL and IC50 (R greater than 0.95). The types of dysmorphogenesis in vitro were those typical for retinoids, and for the most part resembled the malformations found in vivo. We conclude that the whole embryo culture system is a useful tool for the preliminary testing of retinoids.

Abnormalities, Drug-Induced↗
PubMed1992

Comparison of the metabolic activity of yolk sac tissue in the whole embryo and isolated yolk sac culture.

In rat visceral yolk sac tissue cultured from 10.5 to 12.5 days of prenatal age, metabolism of the lipoxygenase inhibitor N-hydroxy-N-methyl-7-propoxy-2-naphtalinethanamine (QA 208-199, QAB) and the accumulation of its metabolites have been shown previously. In this study, the metabolic activities of visceral yolk sac tissues cultured either alone or together with the embryo were compared. The metabolite patterns in medium and visceral yolk sac tissue generated by intact conceptuses or by isolated visceral yolk sac tissues were similar. After 24 as well as 48 h of culture, the major in vivo metabolite, 7-propoxy-naphthalene-2-ylacetic acid (QAA) and other, as yet unidentified metabolites, accumulated in embryo proper and visceral yolk sac tissues. QAB was not found in the embryo proper, and was only found in yolk sac tissues using the higher concentration. In particular the metabolites M5 and M6 exhibited a massive accumulation in the visceral yolk sac, whereas QAA, M3, and M4 accumulated to a much lesser degree. In isolated yolk sacs cultured for 48 h, tissue levels of QAA and M4 were similar to those in yolk sacs of cultured whole conceptuses, whereas M5 and M6 exhibited twofold higher levels in isolated yolk sacs. These findings were in agreement with the distinct increase of myeloid figures containing partly fragmented inclusion bodies in yolk sac tissues. These results suggest that the visceral yolk sac may be the major site of QAB metabolism in cultured rat conceptuses in vitro.

Animals↗
PubMed1991

Effects of QA 208-199 and its metabolite 209-668 on embryonic development in vitro after microinjection into the exocoelomic space or into the amniotic cavity of cultured rat conceptuses.

The objective of this study was to determine the direct embryotoxic effects in vitro of N-hydroxy-N-methyl-7-propoxy-2-naphthalene-ethanamine (QA 208-199, QAB) and of one of its metabolites, 7-propoxy-naphthalene-2-ylacetic acid (209-668, QAA), after circumventing the bioconverting conceptual membranes. The compounds were, therefore, microinjected either into the exocoelomic space or into the amniotic cavity of rat conceptuses of 10 d at prenatal age at doses of up to 84.9 ng (QAA) and 180 ng (QAB) per conceptus respectively. The conceptuses were subsequently cultured for 28 h after which their development was assessed. QAB produced marginal effects on embryonic differentiation only after microinjection of the compound into the amniotic cavity. Dysmorphogenic effects, however, occurred in a dose-dependent fashion after either exocoelomic or intraamniotic microinjections of the compound. The frequencies and types of anomalies were similar after either exposure route and consisted predominantly of anomalies associated with axial rotation. QAA also impaired embryonic differentiation at only the high dose level of 84.9 ng per embryo and after intraamniotic injections only. Dysmorphogenic effects were observed in all experimental groups, although the differences were not statistically significant when compared with the concomitant controls. An increased proportion of anomalies observed were in the cephalic region as compared to the defects produced by QAB. These data suggest that QAA most probably is not the QAB metabolite responsible for the embryotoxic action of QAB in vitro. Furthermore, the results tend to confirm the suggested involvement of the visceral yolk sac membrane in mediating QAB embryotoxicity.

Amnion↗
PubMed1987

Differential susceptibility of immature rat testes to doxorubicin at critical stages of maturation. Biochemical and functional assessment.

The toxic effects of doxorubicin on the reproductive system of the male rat were studied at different susceptible stages of postnatal development. A multidisciplinary approach including the assessment of histopathological, functional and biochemical parameters was chosen. Groups of male rats were treated once with the compound (3 mg/kg) on postnatal day 6, 16, 24 or 45. Both the onset of reproductive capacity and fertility were determined by serially mating ten animals per group for 12 weeks beginning at the age of 45 days. Reproductive organ weights, sperm counts and epididymal androgen binding protein (ABP) were measured at intermediate (80-day-old rats) or terminal sacrifice (129-day-old rats). Age dependent differential doxorubicin toxicity was evident. Treatment of 6-day-old animals with doxorubicin severely impaired development of reproductive functions. Treatment of 16-day-old animals reduced fertility throughout the mating study, as well as body and reproductive organ weights and sperm counts. Initial toxicity was observed in the group treated at 24 days of age; particularly, low reproductive organ weights and low sperm counts were found. These findings proved reversible towards the end of the study. Neither biochemical nor functional impairment of the reproductive system could be observed in the group treated at 45 days of age.

Animals↗
PubMed1985

Testicular toxicity of antineoplastic drugs during postnatal development of the rat.

A multidisciplinary approach was chosen to assess effects of anticancer drugs on the postnatally developing reproductive system. Male rats were treated once with doxorubicin (3 mg/kg bw i. p.) on either postnatal day 6, 16, 24, or 45. Mating studies, sperm counts, estimation of androgen binding protein and assessment of pathological parameters were performed at the time of onset of reproductive capacity of the controls and for 12 weeks thereafter. Male reproductive toxicity of doxorubicin was clearly dependent on the age at the time of treatment, being greatest for 6 day old animals and absent for 45 day old animals.

Androgen-Binding Protein↗
PubMed1984

Aspects of testicular toxicity induced by anticancer drugs.

Newer histopathologic techniques were used in combination with sperm head counts (SHC) and serial mating (SM) studies to assess different aspects of testicular toxicity. Adult male rats were treated once intraperitoneally (i.p.) with vincristine (Vin: 0.15 and 0.6 mg/kg) or procarbazine (Proc: 50 and 200 mg/kg). Investigations were performed at weekly intervals until 10 weeks after treatment. SHC are a good parameter for cytotoxicity (Vin, Proc) assessed in great detail by morphological examination. Optimal fixation after perfusion and high optical resolution of semi-thin sections also allow the study of early and subtle specific alterations. However, they do not replace SM studies for the assessment of genotoxicity (Proc). In turn, SM studies are poor indicators of cytotoxicity.

Animals↗
PubMedDate not supplied

Interlaboratory evaluation of embryotoxicity in the postimplantation rat embryo culture.

The embryotoxicity of eight xenobiotic compounds in rat postimplantation whole embryo culture was blindly tested in four laboratories according to a standard protocol. The results show that the four nonteratogens amaranth, penicillin, isoniazid, and saccharin did not affect embryogenesis apart from general toxicity at very high concentrations in culture for amaranth and isoniazid. There was good concordance of results across the laboratories. The four teratogens (retinoic acid, 6-aminonicotinamide, acetylsalicylic acid, and vincristine) induced a variety of specific embryotoxic effects, which were in most cases similar in all laboratories. These results indicate that the definition for specific embryotoxicity used, as well as the culture duration and embryonic age are crucial for concordant scoring. Other methodologic differences did not significantly influence scoring of embryotoxicity. Therefore, within the limits of the end points and embryonic stage represented in the method, embryo culture appears as a useful method for embryotoxicity screening, which can be reproducibly applied in different laboratories.

Amaranth Dye↗
PubMedDate not supplied

Flow cytometric evaluation of the effects of doxorubicin on rat spermatogenesis.

Histopathologic examination of testicular tissue allows testicular impairment to be investigated. As an alternative to histopathology, flow cytometry (FCM) using a triple staining technique that combines DNA-ploidy with mitochondria stainability and vimentin immunostaining has also been utilized to evaluate testicular damage. In this article we evaluate the effects on spermatogenesis after acute exposure of rats to doxorubicin. Testicular cell suspensions of treated and control animals were analyzed by FCM. This allows several cell types to be identified and quantified, giving a control pattern. Deviations from this control pattern are considered as an indication of testicular damage. Doxorubicin produced a depletion of spermatogonia as early as 3 d after treatment. This effect could be followed through the temporal evolution of spermatogenesis. Comparable results were obtained by histopathology. The presented results show that FCM is a suitable and sensitive method for the detection of testicular damage. The advantages of FCM over other techniques include its rapidity and objectivity.

Animals↗