Biomedical subjects

D L Reimer

Publications and source records attributed to D L Reimer.

At least 19 recordsLinked to original sources

PubMed1999

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

Animals↗

PubMed1997

Analysis of cationic liposome-mediated interactions of plasmid DNA with murine and human melanoma cells in vitro.

Lipid-based DNA transfer formulations are typically selected on the basis of in vitro transfection studies where the activity of specific formulations is defined by transgene expression. It is unclear, however, whether expression is directly related to the efficiency of DNA transfer. In an attempt to correlate DNA transfer with transgene expression, we used a simple assay consisting of measuring DNA (3H-plasmid encoding for beta-galactosidase) binding to murine (B16/BL6) and human (KZ) melanoma cells in vitro at 4 and 37 degrees C. The difference in cell association at these temperatures was assumed to be a consequence of DNA uptake, an assumption that was confirmed by protease removal of cell surface-associated DNA. DNA associated with B16/BL6 melanoma cells (up to 30 ng or 12% of the added DNA) following incubation with dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (DOPE) liposome-DNA aggregates was comparable to that achieved with 1,2-dioleoyloxypropyl-3-trimethylammonium bromide/DOPE or dimethyldioctadecylammonium bromide/DOPE liposomes; however, transgene expression was 2- and 5-fold less for the latter two formulations, respectively. Similarly, equivalent amounts of DNA delivery were achieved with B16/BL6 and KZ melanoma cells, yet the level of transgene expression in the KZ cells was undetectable. It was demonstrated that the lack of transgene expression was not a consequence of cell-specific differences in DNA degradation.

Animals↗

PubMed1997

Self-assembling DNA-lipid particles for gene transfer.

PURPOSE: We have demonstrated that a heteromolecular complex consisting of cationic lipids and DNA can be prepared and isolated (1). Cationic lipids bind DNA through electrostatic interactions. However, when sufficient lipids are bound to DNA the physical and chemical properties of the complex are governed by hydrophobic effects. Here we describe an approach where this hydrophobic complex is used as an intermediate in the preparation of lipid-DNA particles (LDPs). METHODS: The approach relies on the generation of mixed micelles containing the detergent, n-octyl beta-D-glucopyranoside (OGP), the cationic lipid, N-N-dioleoyl-N, N-dimethylammonium chloride (DODAC), and selected zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or egg sphingomyelin (SM). RESULTS: When these micelles were prepared at low detergent concentrations (20 mM OGP) and combined with pCMV beta DNA, LDPs spontaneously formed. The mean diameter of these particles as measured by quasielastic light scattering was 55-70 nm, a result that was confirmed by negative stain electron microscopy. Further characterization of these LDPs showed that DNA within the particles was inaccessible to the small fluorochrome TO-PRO-1 and protected against DNase I degradation. LDPs could also be prepared in high concentrations of OGP (100 mM), however particles formed only after removal of OGP by dialysis. Particles formed in this manner were large (> 2000 nm) and mediated efficient transfection of Chinese hamster ovary cells. Transfection activity was greater when the lipid composition used consisted of SM/ DODAC. Small particles (< 100 nm) prepared of SM/DODAC were, however, inefficient transfecting agents. CONCLUSIONS: We believe that LDP formation is a consequence of the molecular forces that promote optimal hydrocarbon-hydrocarbon interactions and elimination of the hydrocarbon-water interface.

Animals↗

PubMed1996

Cationic lipid binding to DNA: characterization of complex formation.

We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.

Binding Sites↗

PubMed1996

Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes.

Cationic liposomes bound to plasmid DNA are currently used for in vitro and in vivo gene therapy applications, but such complexes readily form large, heterogeneous aggregates that are not appropriate for pharmaceutical development. More importantly, size heterogeneity makes studies focused on optimizing gene transfer to cells difficult to conduct or understand. For this reason we have evaluated the effect of microprobe sonication on these complexes in an effort to achieve process-controlled size homogeneity. Complexes were prepared using a 7.2 kb reporter plasmid and the following liposomal lipid combinations: DDAB/DOPE (50:50 mol %), DDAB/DOPE/PEG-PE (50:45:5 mol %), DDAB/EPC (50:50 mol %), DDAB/EPC/PEG-PE (50:45:5, 50:40:10, 50:35:15 mol %), DODAC/DOPE (50:50 mol %), and DODAC/EPC (50:50 mol %) (DDAB, dimethyldioctadecylammonium bromide; DOPE, dioleoylphosphatidylethanolamine; PEG-PE, monomethoxypolyethylene glycol2000 succinate- distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; DODAC, dioleoyldimethylammonium chloride). The influence of complex composition and lipid:DNA ratio was evaluated. Particle size was determined before and after complexation and again after sonication using the quasi-elastic light scattering technique. DNA integrity was assessed via agarose gel electrophoresis. Finally, gene transfection was evaluated using CHO cells that were transfected in vitro with sonicated and unsonicated complexes. It is established in this study that size reduction can occur, but this is dependent on cationic and neutral lipid composition and, in some cases, lipid:DNA ratio. Surprisingly, the process of sonication leaves a significant percentage of the plasmid DNA intact and capable of in vitro transfection. This study shows that plasmid DNA can be protected from damage due to sonication by liposome complex formation. This may indicate that more common pharmaceutical methods for size reduction which subject particles to mechanical stress may be applicable in preparation of liposome/DNA formulations for in vivo application.

Animals↗

PubMed1996

Distinct mRNA-binding proteins interacting with short repeat sequences of the 3' UTR may be involved in the post-transcriptional regulation of the mouse catalase gene, Cas-1.

The 3' untranslated region (UTR) of the mouse catalase gene (Cas-1) is demonstrated to be an active site for specific protein interactions. We have identified two regions of the Cas-1 3' UTR mRNA that bind to distinct cytoplasmic proteins: one containing a (CA)31 repeat with UA octomer (RNA 5) and another with a (U)15 tract (RNA 6). RNA 5 interacts with one set of protein complexes (a, b, and c) whereas RNA 6 interacts with another (x, y, and z) in a sequence-specific manner. These RNA-protein complexes are development-, tissue-, and genotype-specific. The proteins involved in the two sets of complexes are different. Further characterization of the proteins involved in these interactions has revealed the presence of a single protein of approximately 70 kD that binds RNA 5, and two proteins approximately 38 kD and approximately 47 kD that bind to RNA 6. The approximately 70-k D and approximately 38-kD proteins are also associated with the polysomal fractions and may play a role in the post-transcriptional regulation of Cas-1. Although the observed 3' UTR RNA-protein interactions are hypothesized to be important in post-transcriptional regulation of this gene in rodents, specific RNA sequences and their associated proteins identified in this report would now permit the elucidation of the mechanisms of their action at the molecular level.

Acatalasia↗

PubMed1995

Formation of novel hydrophobic complexes between cationic lipids and plasmid DNA.

An ability to generate a well defined lipid-based carrier system for the delivery of plasmid DNA in vivo requires the characterization of factors governing DNA/lipid interactions and carrier formation. We report that a hydrophobic DNA/lipid complex can be formed following addition of cationic lipids to DNA in a Bligh and Dyer monophase consisting of chloroform/methanol/water (1:2.1:1). Subsequent partitioning of the monophase into a two-phase system allows for the extraction of DNA into the organic phase. When using monovalent cationic lipids, such as dimethyldioctadecylammonium bromide, dioleyldimethylammonium chloride, and 1,2-dioleyl-3-N,N,N-trimethylaminopropane chloride, greater than 95% of the DNA present can be recovered in the organic phase when the lipid is added at concentrations sufficient to neutralize DNA phosphate charge. When the polyvalent cationic lipids 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1- propanaminium trifluoroacetate and diheptadecylamidoglycyl spermidine are used, efficient extraction of the DNA into the organic phase is also achieved when the charge ratio between lipid and DNA is approximately equal. Formation of the hydrophobic DNA complex can only be achieved with cationic lipids. In the absence of added cations or in the presence of excess Ca2+, L-lysine, or poly(L-lysine), 100% of the DNA is recovered in the aqueous fraction. The monovalent cationic lipid/DNA complexes can also be prepared in the presence of detergent; however, low concentrations of NaCl (< 1 mM) lead to dissociation of the complex. Importantly, these results clearly demonstrate that cationic lipid binding does not lead to DNA condensation. The methods described, therefore, enable DNA/lipid complexes to be characterized in the absence of DNA condensation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗

PubMed1994

Complete cDNA and 5' genomic sequences and multilevel regulation of the mouse catalase gene.

Extensive studies over two decades have established strain- and tissue-specific temporal expression of the antioxidant enzyme catalase and have generated an acatalasemic strain of mice C3H/HeAnl/Cas-1b (Csb). This background permits the characterization of the molecular features of the regulation of this important housekeeping enzyme encoded by the gene Cas-1 localized to chromosome 2. We report the first complete cDNA sequence for Cas-1, including 659 bp of the 5' genomic upstream regulatory region. Cas-1 expression (e.g., mRNA, polypeptide, and enzyme activity) in tissues from appropriate strains is evaluated. The genotype- and strain-specific differences in tissue expression appear to be post-transcriptionally regulated. The mRNA stability is unaffected, and the regulation must involve translational efficiency or post-translational protein stability. The TATA-less 5' promoter is CG rich, with potential tissue-specific differences in methylation. The 3' UTR has unusual repeats [(CA)31, (T)15, (TGTGC)7] and may form mRNA-protein complexes. Here, 3' UTR binding protein(s) may account for transacting factors recognized in segregation studies. We propose that this housekeeping antioxidant enzyme is under multilevel regulation, and the determinants include both 5' and 3' Cas-1 sequences.

Amino Acid Sequence↗

PubMed1990

In situ hybridization studies on murine catalase mRNA expression during embryonic development.

In situ hybridization using nucleic acid probes was used to detect cell- and tissue-specific transcript(s) of embryonic genes during development and differentiation. This highly sensitive technique has the potential to provide valuable information on the regulation of low-abundance housekeeping genes during development. We have determined the experimental conditions required to detect the catalase message in adult mouse liver. Catalase effects the breakdown of H2O2 to O2 and H2O and offers protection against the toxic effects of oxygen radicals. We used a cloned 550 bp BamHl-Pstl fragment from a mouse catalase cDNA (pMCT-1) to generate 35S-labeled sense and antisense riboprobes. The experimental conditions used were sensitive enough to quantitate the abundance of silver grains generated by the antisense riboprobe on the adult liver, a tissue known to be positive for this message. The hybridization protocol was applied to serial sections of 13- and 18-day-old mouse embryos. The results suggest that the catalase expression in the liver and brain begins with somite formation and increases with development and differentiation. On the other hand, this message appears to be absent in mesenchyme, particularly in day 13 embryos. The message in positive tissues appears evenly distributed throughout the cell. The observed expression of the catalase message in the adult liver is approximately six times that in the embryonic liver. It is compatible with the enzyme activity results and emphasizes the sensitivity of the in situ hybridization method (over northern blot, etc.) used in this study.

Animals↗

PubMed1989

Pathogenesis of experimental rabies in mice: an immunohistochemical study.

The spread of rabies virus in the central nervous system of mice was examined after hindlimb footpad and intracerebral inoculation of the CVS strain of fixed rabies virus. All mice developed paralytic rabies. After intracerebral inoculation there was early simultaneous infection of neurons in the cerebral cortex and pyramidal neurons of the hippocampus, and later there was spread to the cerebellum. After high-dose intracerebral inoculation there was early infection in ependymal cells lining the lateral ventricles and neurons adjacent to the central canal of the spinal cord, suggesting that rabies virus entry into the CNS occurs, at least in part, by a cerebrospinal fluid pathway. The sequence of involvement was different after hindlimb footpad inoculation. Infection became established in the cerebellum on day 5, in the cerebral cortex on day 6, and in the hippocampus on day 8. CA3 was initially affected, CA1 became infected 2 days later, and there was much less involvement of the dentate gyrus. Hippocampal infection occurred late relative to the rest of the brain after peripheral inoculation, but not after intracerebral inoculation. The hippocampus is not a good location for the detection of early brain infection after peripheral inoculation, although it may be involved when a natural rabies vector has the ability to transmit infection. These findings also raise questions about the mechanisms for the limbic dysfunction observed in clinical rabies.

Animals↗

PubMed1989

Detection of rabies virus RNA in the central nervous system of experimentally infected mice using in situ hybridization with RNA probes.

Rabies virus is usually demonstrated in human or animal tissues using antigen-detection or viral isolation techniques. Rabies virus RNA can be demonstrated in paraffin-embedded tissues using in situ hybridization. Negative (-) sense 35S- and 3H-labeled RNA probes, specific for rabies virus nucleocapsid protein mRNA, were used for the detection of rabies virus RNA in the nervous system of mice experimentally infected with fixed and street strains of rabies virus. In situ hybridization signals were compared with rabies virus antigen demonstrated with immunoperoxidase staining. Rabies virus RNA and antigen were also demonstrated in the same neurons using a double-labeling technique. In situ hybridization has potential applications as a diagnostic test for rabies and in studies of rabies pathogenesis.

Animals↗

PubMed1987

Evidence for unequal crossing-over as the mechanism for amplification of some homogeneously staining regions.

The mechanism of DNA amplification in homogeneously staining regions (HSR) was studied in the human melanoma cell line MeWo. Three karyotypically distinguishable cell types within this cell line contain HSR on four different chromosomes, but all HSR contain the same amplified sequences derived from the short arm of chromosome #15. We examined metaphases of MeWo cells from different passages for changes in the length and location of the HSR. In addition, we examined the replication patterns of the HSR sequences and the organization of repeated sequences within these structures. We found that variation in the lengths of the HSR was due to fewer or more copies of a unit that consisted of a later-replicating, distamycin A/DAPI-positive block and active nucleolar organizing regions (NOR). Lateral asymmetry studies suggested that the satellite DNA sequences that are present within the HSR are organized in large inverted repeats. This organization would account for the pairing in both orientations with exchanges resulting in the types of derivative chromosomes observed. The frequency of sister chromatid exchanges (SCE) within the HSR was increased over other chromosomal regions and four examples of unequal SCE within the HSR, with prominent looping out of the longer chromatids, were seen. These results support a model of unequal SCE, rather than saltatory replication for the amplification of DNA sequences in these HSR.

Cell Line↗

PubMed1986

Rearrangements of chromosomal regions containing ribosomal RNA genes and centromeric heterochromatin in the human melanoma cell line MeWo.

A chromosomal examination of cells from the earliest available passage of the human melanoma cell line MeWo revealed the presence of seven hypodiploid cell types that shared common complex marker chromosomes. Two of the cell types had long homogeneously staining regions (HSR) by Q-banding on three different chromosomes. Distamycin A/DAPI staining and silver staining for active nucleolar organizing regions (NOR) confirmed that the HSR were derived from chromosome #15. All HSR-containing cells had 4-9 pairs of large NOR distributed along the length of each HSR, with all acrocentric chromosomes being negative. The HSR-lacking cells differed primarily with respect to the morphology of the short arm of one #13 chromosome and NOR activity. One cell type had four chromosomes with active NOR, whereas all other cell types had a single active NOR on one #13. One of these cell types had a satellited #8 with NOR. Cells from three other MeWo cultures at higher passages were examined. Two of these contained both hypodiploid and hypotetraploid cells, some of which had satellited X chromosomes or satellited #3 chromosomes with active NOR. The majority of the new chromosomal rearrangements in cells from the later cultures involved the NOR-containing regions, many of which were associated with the distamycin A/DAPI-positive centromeric heterochromatin from chromosome #15. These results indicate that the chromosomal instability in the MeWo cultures is mainly limited to sequences containing active NOR and centromeric heterochromatin from chromosomes #13 and #15. This may be due to a selective pressure to increase the number of active NOR in the MeWo cells. If this is so, it would appear that amplification of active NOR occurs more readily than the activation of the many silent NOR present in these cells.

Cell Line↗

PubMed1985

Amplified sequences from chromosome 15, including centromeres, nucleolar organizer regions, and centromeric heterochromatin, in homogeneously staining regions in the human melanoma cell line MeWo.

Homogeneously staining regions (HSRs) in the human melanoma cell line, MeWo, are located on an X and a der(15) chromosome. These regions are homogeneously stained with quinacrine fluorescence, but stain differentially with conventional Giemsa, G-banding, C-banding, and distamycin A/4',6-diamidino-2-phenylindole. There are five and six blocks of positively staining material on the X and der(15) HSRs, respectively. Hybridization in situ with a cloned repetitive Kpn I family member has confirmed the amplification of this sequence along the HSRs. With silver staining of the nucleolar organizer regions (NORs) there appear to be three very strong and two weaker pairs of NORs along the HSR of the X chromosome and four strong and one weaker pair on that of the der(15) chromosome. There was little NOR staining on the normal acrocentric chromosomes in these cells, suggesting preferential transcription of the NORs in the HSRs. Centromere-dot staining revealed a distribution of multiple centromeres similar to the NORs along the two HSRs. These data suggest that a unit composed of the short arm and centromere of chromosome #15 has been amplified and that the HSR present on the X chromosome probably arose by a translocation from chromosome #15.

Cell Line↗

PubMed1985

DNA amplification and tumorigenicity of the human melanoma cell line MeWo.

Homogeneously staining regions (HSRs) were found in hypodiploid cells (40%) of a subline of the human melanoma cell line, MeWo, (MeWo-C) but were absent from the hypotetraploid cells (60%). Another subline (MeWo-B) was also shown to contain two populations of cells, 70% hypodiploid and 30% hypotetraploid. None of the MeWo-B cells contained HSRs, but all four cell types from both sublines shared marker chromosomes indicating their common origin. The hypodiploid MeWo-B cells were karyotypically similar to the hypodiploid MeWo-C cells except for the presence of the HSRs in the latter. Both MeWo-C and MeWo-B sublines were injected into BALB/c nude mice. The MeWo-C cells were markedly more tumorigenic than MeWo-B cells as judged by tumor incidence, latency, average tumor size, and tumor take values. Cytogenetic and flow cytofluorometric analyses of the tumors induced by MeWo-C cells revealed a shift in the tumor cell population from 40% to greater than 90% HSR-containing hypodiploid cells during tumor growth. Hybridization of tumor DNA to a probe (D15Z1), the sequence of which is amplified in the HSRs, also indicated an increase in the proportion of HSR-bearing cells during tumor growth. No such selective advantage was found with the hypodiploid, HSR-lacking MeWo-B cells. The results suggest that HSRs found in the human melanoma line MeWo may confer enhanced tumorigenicity to the cells containing them.

Animals↗

PubMed1984

Distribution of sister chromatid exchanges on the mouse chromosomes in vivo with reference to the replication properties of the X chromosome.

Frequency of sister chromatid exchanges (SCE) were recorded separately for different chromosomes from bone marrow cells of female mice of the two genetic strains (C3H/S and C57BL/6J). SCEs were evaluated following different doses of 5-bromo-2'-deoxyuridine (BrdU) as nine hourly i.p. injections. The SCE per cell increased with increasing BrdU doses which was slightly higher in C3H/S than in the C57BL/6J. SCEs per cell were variable at every treatment-strain combination, possibly reflecting the heterogeneous nature of the bone marrow cells. In general, there is a positive correlation between SCE per chromosome and the relative chromosome length. Total SCEs on one of the large chromosomes (most likely the X chromosome), however, are significantly higher than expected on the basis of relative length alone. Most of this increase is attributable to one of the homologues of this chromosome, which is not in synchrony with the rest of the chromosomes and may represent the late-replicating X. These results when viewed in the light of replication properties of the heterochromatinized X, suggest a direct involvement of DNA replication in SCE formation and may argue against the replication point as the sole site for the SCEs.

Animals↗

PubMed1983

Cyclophosphamide-induced in vivo sister chromatid exchange in Mus Musculus. II: Effect of age and genotype on sister chromatid exchange, micronuclei and metaphase index.

In vivo cyclophosphamide-induced sister chromatid exchanges (SCEs) micronuclei, and metaphase indices were assessed in two age groups (10.8 +/- 0.9 weeks' an 33.1 +/- 1.3 weeks' old) of female mice from three genetic strains (C3H/S, C57BL/6J, and Balb/c). In general, older animals showed diminished SCE induction over their younger counterparts. The relative difference between individuals of the two ages is strain-dependent. Unlike C57BL/6J and Balb/c, strain C3H/S showed significantly lower SCE values in the older animals at every cyclophosphamide treatment. It may reflect on the possible involvement of genetic determinant(s) for the component(s) of SCE formation during aging. Frequencies of micronuclei, however, were consistently higher in older animals than in their younger counterparts. Furthermore, cytotoxicity of cyclophosphamide, as reflected in metaphase indices, was also higher in older animals. Lower metaphase indices associated with higher micronuclei levels in older individuals may suggest a decline in the rate of cellular replication in these animals. Furthermore, the lower metaphase indices associated with lower SCE values, and increasing micronuclei levels accompanied by decreasing SCE frequencies in older animals, may reflect reduced DNA repair ability during aging. These results support the hypothesis of genotype-dependent decline in the rate of DNA repair and replication during aging, particularly under stressed conditions.

Aging↗

PubMed1983

Cyclophosphamide-induced in vivo sister chromatid exchanges (SCE) in Mus musculus. III. Quantitative genetic analysis.

In vivo cyclophosphamide (CP)-induced sister chromatid exchanges (SCEs) were evaluated in females from five genetic strains of mice (C57BL/6J, C3H/S, 129/ReJ, BALB/c and DBA/2) and their F1 hybrids. Baseline (noninduced) SCE values differ significantly among strains, 129/ReJ having the lowest and DBA/2 having the highest mean SCE per cell values. In general, the baseline SCE of a given F1 is within the range of its corresponding parental strains or near the lower parental value. Furthermore, there is a genotype-dependent increase in mean SCEs per cell with CP dose. Strain differences in SCE induction are noted particularly at the two higher CP doses (4.50 and 45.0 mg/kg). In general, F1 hybrids involving a strain with high induced SCEs and a strain with low induced SCEs exhibit mean SCE values that are closer to the value of the lower strain. F1s involving two strains with high SCEs or two strains with low SCEs yield SCEs not different from parental strains. The method of diallel cross analysis showed the order of dominance of these strains in SCE induction to be 129/ReJ BALB/c C3H/S DBA/2 C57BL/6J. These results support the involvement of predominantly nonadditive genetic factors as major gene(s) in SCE induction. In addition, involvement of random and independent events in SCE induction is suggested by the distribution of SCEs which follows a Poisson distribution.

Animals↗