Unusual cytoskeletal and chromatin configurations in mouse oocytes that are atypical in meiotic progression

Meiotic maturation progresses atypically in oocytes of strain LT/Sv and l/LnJ mice. LT/Sv occytes show a high frequency of metaphase l-arrest and parthenogenetic activation. l/LnJ oocytes display retarded kinetics of meiotic maturation and a high frequency of metaphase l-arrest. Some l/LnJ oocytes fail to resume meiosis. Changes in the configuration of chromatin, microtubules, and centrosomes are associated with specific stages of meiotic progression. In this study, the configuration of these subcellular components was examined in LT/Sv, l/LnJ, and C57BL/6J (control) oocytes either freshly isolated from large antral follicles or after culture for 15 hr to allow progression of spontaneous meiotic maturation. Differences were found in the organization of chromatin, microtubules, and centrosomes in LT/Sv and l/LnJ oocytes compared to control oocytes. For example, rather than exhibiting multiple cytoplasmic and nuclear centrosomes as in the normal germinal vesicle-stage oocytes, LT/Sv oocytes typically contain a single large centrosome. In contrast, l/LnJ oocytes displayed many small centrosomes. The microtubules of normal germinal vesicle-stage oocytes were organized as arrays or asters, but microtubules were shorter in LT/Sv oocytes and absent from l/LnJ oocytes. After a 15-hr culture, centrosomal material of normal metaphase II oocytes was organized at both spindle poles. In contrast, metaphase l-arrested LT/Sv oocytes exhibited an elongated spindle with centrosomal material appearing more organized at one pole of the spindle. Both control and LT/Sv oocytes displayed cytoplasmic centrosomes. Metaphase l-arrested l/LnJ oocytes rarely had cytoplasmic centrosomes but exhibited centrosomal foci at the spindle periphery. Thus, oocytes that are atypical in the progression of meiotic maturation displayed aberrant configurations of microtubules and centrosomes, which are thought to participate in the regulation of meiotic maturation.     

Report from the in vitro micronucleus assay working group

At the Washington “2nd International Workshop on Genotoxicity Testing” (25–26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth “3rd International Workshop on Genotoxicity Testing” (28–29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern:

1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test.

2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information.

3. Treatment schedules for cell lines and lymphocytes.

4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly).

5. Duplicate cultures and number of cells to be scored.

6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative.

7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.

     

Molecular analyses of a Lesch-Nyhan syndrome mutation (hprt Montreal) by use of T-lymphocyte cultures

Summary The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (TC transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine₅₆. We have designated this new Lesch-Nyhan mutation hprt _Montreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.     

Betulaceae, Corylaceae, Cupressaceae, Fagaceae and Salicaceae around Parma (Northern Italy): Pollen calendars from 1995 to 1997

Many seasonal respiratory allergies are caused byairborne pollens. There is an evident correlationbetween allergic attacks and the amount of pollens inthe atmosphere at any time. The airbornetree pollen concentration and the relevantrepercussions on pollinotics hasnot yet been extensively reported for all Italian regions.We present the results of a 3-year, weekly, tree pollen count byBurkard spore trap from the atmosphere of Parma,Italy. Annual pollen calendars were made from theresults. The study has been conducted because, in Europe,an increasing frequency of allergic sensitisation to thesepollens has been observed.     

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.     

Formation of copper-bleomycin complexes: evidence of a three-step process

The formation of Cu(II)-bleomycin complexes as a function of pH has been studied using circular dichroism, absorption, electron paramagnetic resonance spectroscopy, and potentiometric titration. Our data support the following points: the formation of Cu(II)-bleomycin complexes occurs in a three-step process: a first complex (I) is formed at pH 1.2, which most probably involves the pyrimidine nitrogen, the secondary amine nitrogen, and two water molecules as the four in-plane ligands of copper. A second complex (II) is formed at pH 2.5, through the further coordination of the peptide nitrogen of histidine residue, and histidine imidazole nitrogen giving rise to the release of two protons. The fixation, in apical position, of the alpha-amino nitrogen of beta-aminoalanine occurs in a last step through the release of one additional proton. A value of 2.7 has been obtained for the pK of formation of this third complex, which is the species present at physiological pH. In the Cu(II)-depbleomycin system only one complex (II') has been detected.