Clinical and anatomic pathology effects of serial blood sampling in rat toxicology studies, using conventional or microsampling methods.

UNLABELLED: As a general practice in rodent toxicology studies, satellite animals are used for toxicokinetic determinations, because of the potential impact of serial blood sampling on toxicological endpoints. Besides toxicological and toxicokinetic determinations, blood samples obtained longitudinally from a same animal may be used for the assessment of additional parameters (e.g., metabolism, pharmacodynamics, safety biomarkers) to maximize information that can be deduced from rodents. We investigated whether removal of up to 6 × 200 µL of blood over 24h can be applied in GLP rat toxicology studies without affecting the scientific outcome. METHODS: 8 week-old female rats (200-300 g) were dosed for up to 1 month with a standard vehicle and subjected or not (controls) to serial blood sampling for sham toxicokinetic/ancillary determinations, using miniaturized methods allowing collection of 6 × 50, 100 or 200 µL over 24h. In-life endpoints, clinical pathology parameters and histopathology of organs sensitive to blood volume reduction were evaluated at several time points after completion of sampling. RESULTS: In sampled rats, minimal and reversible changes in red blood cell mass (maximally 15%) and subtle variations in liver enzymes, fibrinogen and neutrophils were not associated with any organ/tissue macroscopic or microscopic correlate. CONCLUSION: Serial blood sampling (up to 6 × 200 µL over 24h) is compatible with the assessment of standard toxicity endpoints in adult rats.

Inhibition of iPLA2 ß and of stretch-activated channels by doxorubicin alters dystrophic muscle function.

BACKGROUND AND PURPOSE: Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice. EXPERIMENTAL APPROACH: Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. KEY RESULTS: Dox at 1-30 µM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. CONCLUSIONS AND IMPLICATIONS: Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

Spontaneous metritis related to the presence of vaginal septum in pregnant Sprague Dawley Crl:CD(SD) rats: impact on reproductive toxicity studies.

Recently, 6% of 1,176 Sprague Dawley rats examined in our reproductive toxicity studies presented with dark-red uterine contents with or without fetuses demonstrating delayed development. Sometimes, a high proportion of the litter was found dead, and dystocia with death or preterminal euthanasia of the dam occurred. Microscopic findings in the uterus consisted of necrohemorrhagic and suppurative periplacentitis associated with the presence of bacterial colonies identified as Escherichia coli. In the vagina, similar findings were observed that were associated with mucus accumulation and the presence of a transverse occlusive or partially occlusive thin membrane identified as a vaginal septum. Microscopically, this septum consisted of a thin band of connective tissue covered on both sides by a mucous epithelium that was continuous with vaginal epithelium. In some cases, there was only mucus accumulation retained by a septum in the vagina without evidence of bacterial infection. Serological and histological examinations did not reveal any specific pathogenic agent. The presence of these septa in the vagina most likely favored mucus accumulation, nonspecific ascending bacterial infection, and dystocia. This colony of rats presented with an unusually high incidence of vaginal septa as it was described in different strains of mice and rats in the past. We hypothesized that the use of an impedance meter by the breeder--to determine the phase of the estrous cycle by introducing a probe in the vagina--likely facilitated gestation by perforating the vaginal septum in some cases.

Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy.

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP. EXPERIMENTAL APPROACH: The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx(5Cv) mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg(-1)), CsA (10 mg kg(-1)) or placebo. The effects on muscle necrosis and function were measured. KEY RESULTS: In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg(-1), respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg(-1) relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress. CONCLUSIONS AND IMPLICATIONS: Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients.

Induction of DNA synthesis in mouse liver following increases of DNA adduct levels elicited by very low cumulative doses of the genotoxic hepatocarcinogen 7H-dibenzo[c,g]carbazole.

The purpose of this work was to investigate the administration of very low but repeated doses of a genotoxic carcinogen and an eventual correlation with cellular DNA synthesis. The compound 7H-dibenzo[c,g]carbazole is a genotoxic carcinogen in the mouse liver and was administered topically at the dose of 13.35 microg per animal every 2 days to give a total of 13 applications. Animals were sacrificed 48 hours after every 2 applications until the 10th treatment, then 48 hours after every treatment. Postulated genotoxic effects such as DNA adduct formation were detected by the 32P-post labeling assay. Liver sections were examined for microscopic changes and DNA synthesis. Results showed an increase of the total DNA adduct level in the liver throughout the study with a slowing down in the level after the sixth application of the compound. This change could correspond to the onset of DNA synthesis and to the moderate hepatocellular apoptosis which was observed. The DNA synthesis, which was considered to be secondary to the cytotoxicity induced by the high level of DNA adducts altering normal cellular activity, could also be the opportunity to fix the DNA adducts into heritable mutations. These results raise the question regarding the risk assessment in humans exposed regularly to very low doses of chemicals in the environment: for non-proliferating tissue, the regular accumulation of DNA adducts could remain silent until a "threshold level" is reached from which stimulation of the DNA synthesis may fix the DNA adducts into heritable mutations, eventually leading to tumors.

Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system.

To study the pathogenesis of X-linked recessive myotubular myopathy (XLMTM), we used a nerve-muscle coculture system which allows the reconstitution of functional motor units in vitro after coupling of human skeletal muscle cells with embryonic rat spinal cord explants. We used three skeletal muscle cell lines derived from subjects with known mutations in the MTM1 gene (two from embryonic tissues, associated with mutations predicted to give a severe phenotype, and one from a neonate still alive at 3 years 6 months and exhibiting a mild phenotype). We compared these three XLMTM muscle cell cultures with control cultures giving special attention to behaviour of living cocultures (formation of the myofibres, contractile activity, survival), expression of muscular markers (desmin, dystrophin, alpha-actinin, troponin-T, myosin heavy chain isoforms), and nerve-muscle interactions (expression and aggregation of the nicotinic acetylcholine receptors). We were unable to reproduce any 'myotubular' phenotype since XLMTM muscle cells behaved like normal cells with regard to all the investigated parameters. Our results suggest that XLMTM muscle might be intrinsically normal and emphasize the possible involvement of the myotubularin-deficient motor neurons in the development of the disease.