Changes in clinical pathology parameters during gestation in the New Zealand white rabbit.

Hematology and serum chemistry parameters were analyzed in 2 groups of pregnant rabbits to assess changes in these parameters over the course of gestation. These data were used to generate a historical control reference range for embryofetal development regulatory toxicology studies. During the 28-day gestation period, the following major changes were observed. Red blood cell counts, hemoglobin, and hematocrit increased slightly up to day 13 and subsequently decreased progressively to a nadir for all parameters on day 25. Reticulocyte counts increased maximally by day 16 and then decreased to a minimum value on day 28. White blood cell counts progressively declined after day 7. Platelet counts increased slightly by day 10, were relatively stable until day 13, then progressively decreased to a nadir on day 25. Aspartate aminotransferase and alanine aminotransferease values increased steadily throughout the study to reach a maximum value on day 25. Triglycerides increased to their maximum value by day 19 and then steadily decreased until day 28, whereas cholesterol decreased progressively to reach a nadir on day 25. Urea and total protein decreased steadily from day 13 onward. Calcium values decreased throughout the study to reach a minimum value on day 28. Phosphorus values increased slightly on days 7 and 13 and then progressively decreased to reach a nadir on day 28. With a few exceptions, changes that occur in clinical pathology parameters during pregnancy in the rabbit are similar to those observed in pregnant women. Therefore, the rabbit can be considered a suitable species for embryofetal development toxicity studies with regard to clinical pathology.

Comparison of the potency of glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factors in neutropenic and nonneutropenic CD rats.

Recent studies in human bone-marrow culture and healthy human volunteers suggest that lenograstim [glycosylated, recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Chinese hamster ovary (CHO) cells] has greater in vivo potency than filgrastim [nonglycosylated, methionine-extended recombinant human granulocyte colony-stimulating factor (rmetHuG-CSF) produced in Escherichia coli]. To confirm and extend these results we investigated the in vivo potency of both products in normal rats and neutropenic CD rats as an animal model of chemotherapy-induced neutropenia. In normal rats, groups of eight normal male CD rats received four subcutaneous doses of 10, 30, or 100 micrograms/kg filgrastim or lenograstim on days 1-4 of the study, whereas a control group received the vehicle. Blood samples were collected from each animal before treatment (day -5) and on days 2, 3, 5, 8, and 12 of the study for determination of red blood cell (RBC), platelet, white blood cell (WBC), and differential counts. rHuG-CSF and r-metHuG-CSF produced increased WBC counts, principally due to elevated absolute neutrophil counts (ANCs); on days 2, 3, and 5, all groups receiving rG-CSF had ANCs that increased in a progressive and dose-related manner. With the exception of a single value, mean ANCs obtained on days 2, 3, and 5 in lenograstim-treated groups were higher (statistically significant on day 3 at 30 and 100 micrograms/kg and on day 5 at 10, 30, and 100 micrograms/kg) than the respective values obtained in filgrastim-treated groups. No compound-related effect was noted in RBC or platelet parameters. Neutropenia was induced in male CD rats (12 animals/group) with a single intraperitoneal dose of 50 mg/kg cyclophosphamide (CPA) on day 0. On days 1-4, CPA-treated groups were treated with the vehicle (control) or with filgrastim or lenograstim at 30 or 100 micrograms/kg per day. An additional group was not treated with CPA and served as the absolute control group. Blood was collected from alternating subgroups on study day -5 (pretest) and on days 2, 3, 4, 5, 6, 8, 9, and 12 for determination of RBC, platelet, WBC, and differential counts. No major adverse in-life effect was noted in neutropenic rats. Maximal depression of WBCs and ANCs occurred on day 5, followed by recovery to normal values by days 9 (ANC) and 12 (WBC). On day 3 and days 5-9, rHuG-CSF- and metHuG-CSF-treated groups had marked and dose-related increases in WBCs as compared with CPA-treated controls, principally due to elevated ANCs. With the exception of a few values, mean ANC values obtained in lenograstim-treated groups were consistently higher than the respective values obtained in filgrastim-treated groups; the difference was statistically significant on day 3 (30-microgram/kg groups) and on days 6 and 8 (100-microgram/kg groups). In conclusion, treatment of normal and neutropenic CD rats with lenograstim resulted in a dose-related elevation of ANCs that was consistently and significantly higher than the response to identical doses of filgrastim. These results suggest that lenograstim, the glycosylated form of rG-CSF, has superior in vivo potency in normal and neutropenic animals as compared with filgrastim, the nonglycosylated form of rG-CSF.

Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed in E. risticii-containing vacuoles in untreated cells, while significantly greater P-L fusion with ehrlichia-containing vacuoles was observed after oxytetracycline treatment. When latex beads were introduced into uninfected cell cultures, P-L fusion was observed with vacuoles containing latex. Fusion of lysosomes with latex-containing vacuoles in cells was significantly greater than fusion of lysosomes with ehrlichia-containing vacuoles in the same infected cells. These findings indicate that E. risticii is able to inhibit P-L fusion, whereas oxytetracycline deprives organisms of this ability.

Variable hepatocellular vacuolization associated with glycogen in rabbits.

Marked variation in hepatocellular vacuolization was present in New Zealand white rabbits used as controls in 28-day and 91-day percutaneous studies conducted at 5 different laboratories. Vacuoles in hepatocytes of alcohol-fixed and formalin-fixed livers contained periodic acid-Schiff (PAS)-positive material which was removed with diastase digestion, indicating the presence of glycogen. The magnitude of hepatocyte vacuolization was subjectively assessed by light microscopy using 5 histologic grades. Quantitative measurements of hepatocyte perimeter and lobule radius for representative liver sections of each histologic grade corroborated the different grades used. Factors associated significantly with the degree of vacuolization were sex (females were affected more severely than males), body weight, relative liver weight, and the laboratory conducting the study. Also apparent were variations in mean severity of hepatocyte vacuolization between studies conducted at the same laboratory, and variation in severity of vacuolization within individual studies. Duration of the study and season had no significant association with the degree of vacuolization. Marked variation of hepatocellular vacuolization due to glycogen accumulation must be recognized when evaluating results of toxicity testing in rabbits.