Comparison of single or multiple intratracheal administration for pulmonary toxic responses of nickel oxide nanoparticles in rats.

OBJECTIVES: In this study, we focused on the qualitative and quantitative differences of the lung lesions induced by single or multiple intratracheal administration of nickel oxide nanoparticles (NiO). METHODS: Male rats were randomized into groups receiving intratracheal administrations in a single dose or two to four divided doses of 2 mg/kg/bw. Bronchoalveolar lavage fluid (BALF) analyses were performed at 3 and 28 d post-dose. Histopathological analyses were performed at 28 and 91 d post-dose. RESULTS: BALF analyses revealed pulmonary injury, inflammation, and increases in the parameters indicating processing the foreign material in all the NiO-treated groups. Histopathological analyses showed the phagocytosis of NiO by alveolar macrophages, degeneration and necrosis of alveolar macrophages, and inflammatory responses. In the comparison between single and multiple administrations, the trend for stronger toxicity effects was observed after multiple application at 3 d post-dose, while the obvious toxicity effects were also seen in case of single administration. No particular differences of lung lesions depending on the frequency of administration at 28 and 91 d post-dose were evident. CONCLUSION: Intratracheal NiO administration induced strong toxic response thoroughly even by single administration. Therefore, single administration was concluded to be applicable to assess the inhalation toxicity of nanomaterials and can be used in the screening test.

Baseline serum cardiac troponin I concentrations in Sprague-Dawley, spontaneous hypertensive, Wistar, Wistar-Kyoto, and Fisher rats as determined with an ultrasensitive immunoassay.

Cardiac troponins have proved to be reliable blood biomarkers for identifying a variety of myocardial alterations in humans and animals. Recently, an ultrasensitive cTnI assay (Erenna IA) has been used to demonstrate increases in baseline cTnI resulting from drug-induced myocardial injury in rats, dogs, and monkeys, as well as to document baseline cTnI ranges in Sprague-Dawley (SD) rats. The present study was initiated to use the Erenna cTnI assay to further document baseline cTnI concentrations in normal control animals from multiple strains, including SD, Spontaneous Hypertensive (SHR), Wistar, Wistar-Kyoto (WKY), and Fisher strains. Baseline cTnI concentrations were quantified in all rats tested, and males had higher mean cTnI concentrations than females of the same strain. SHR males had the highest mean cTnI concentrations and the largest cTnI variability. Interestingly, cTnI concentrations increased in castrated SHR compared with unaltered male SHR, whereas cTnI concentrations decreased in ovariectomized SHR compared with unaltered female SHR. These results show significant differences in cTnI concentrations between strains, sexes, and noncardiac surgical alterations in control animals, and identify these as potential contributing factors to cTnI baseline variability that should be taken into account when using ultrasensitive cTnI as a biomarker to assess preclinical cardiotoxicity.

Development of a physiologically based pharmacokinetic model for chlorobenzene in F-344 rats.

A physiologically based pharmacokinetic (PBPK) model to describe the absorption, distribution, metabolism, and elimination of chlorobenzene in rats was developed. Partition coefficients were experimentally determined in rat tissues and blood samples using an in vitro vial equilibration technique. These solubility ratios were in agreement with previous reports. The in vivo metabolism of chlorobenzene was evaluated using groups of three F344 male rats exposed to initial chlorobenzene concentrations ranging from 82 to 6750 ppm in a closed, recirculating gas uptake system. An optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants, K(m) (affinity) and Vmax (capacity), using the PBPK model. At the highest chamber concentration, the uptake curve could not be modeled without the addition of a first-order (Kfo) metabolic pathway. Pretreatment with pyrazole, an inhibitor of oxidative microsomal metabolism, had no impact on the slope of the uptake curve. The completed PBPK model was evaluated against real-time exhaled breath data collected from rats receiving either an intraperitoneal (i.p.) injection or oral gavage dose of chlorobenzene in corn oil. Exhaled breath profiles were evaluated and absorption rates were determined. Development of the chlorobenzene PBPK model in rats is the first step toward future extrapolations to apply to humans.

Hypothalamic leptin resistance is associated with impaired leptin signal transduction in aged obese rats.

Leptin contributes to the regulation of both food intake and energy expenditure. We previously demonstrated that the F344xBN rat, a rodent model for late-onset obesity, is leptin-resistant and that leptin signal transduction following peripheral administration of leptin is impaired in these aged, overweight rats. To determine if leptin signal transduction is impaired in response to central administration of leptin and whether reduced hypothalamic leptin receptors may be contributing to the impaired signal transduction, we examined the in vivo dose-response leptin-induced STAT3 activation (phosphorylation and binding activity to the SIE M67 oligonucleotide) in response to i.c.v. administration of leptin along with the level of hypothalamic leptin receptor protein in young and older, late-onset obese rats. The leptin-induced maximum phosphorylation of STAT3 was 41% greater in young compared with older obese rats, but the dose required for half-maximal phosphorylation of STAT3 was similar in both the young (41 ng) and old-obese (47 ng) rats. There were no changes in total STAT3 protein with leptin or age, and leptin did not increase phosphorylation of STAT1. Leptin increased phosphorylation of STAT3 transcription factor binding eight-fold in the young but only four-fold in the aged-obese rats, and leptin receptor protein was 50% greater in the young compared with aged rats. These data indicate that aged-overweight rats demonstrate reduced signal transduction in response to centrally administered leptin that may be the result of the diminished leptin receptor protein observed in the aged-obese rats. The diminished leptin receptors and impaired leptin signal transduction may explain the diminished physiological responses observed following leptin administration in older rats. This impaired leptin signal transduction may be due either to the elevated obesity with age or to age itself, or to both.

Identification of a quantitative trait locus influencing plasma insulin levels after 70% pancreatectomy using the OLETF rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for proliferation of pancreatic beta-cells after partial pancreatectomy, which may be the critical pathogenetic event in NIDDM development. The poor pancreatic beta-cell proliferation in this model is characterized by reduction in beta-cell mass and decrease in insulin content in the remnant pancreas. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for beta-cell mass and plasma insulin levels after partial pancreatectomy by performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We have identified a suggestive QTL for the plasma insulin levels, near D20Mgh5 on rat chromosome 20, with a maximum lod score of 3.75 which accounts for 20% of the total variance, while no QTLs were detected for beta-cell mass. This chromosome 20 QTL, whose OLETF allele is associated with low plasma insulin levels through acting in an incompletely recessive manner, may affect insulin secretion itself rather than beta-cell proliferation.

Corticosterone responses of adult Lewis and Fischer rats.

Indwelling catheters were implanted into the inferior vena cava of adult male and female Lewis/N and Fischer 344 rats. Each animal was exposed to ACTH, novelty stimulation, nicotine, lipopolysaccharide (LPS), and saline on 5 consecutive days. Blood was withdrawn before (baseline) and at several time points after the stimulus on each day. There were no differences in baseline corticosterone levels nor in responses to saline in any group. In general, responses to stimulation peaked at 15-30 min and returned to baseline by 60-90 min. Corticosterone responses to LPS showed a different time course; maximal responses occur at 1-2 h and return to baseline by 24 h. Fischer animals showed higher corticosterone levels than Lewis rats during the response to stimulation, but returned to baseline at the same times. Females of each strain showed higher corticosterone responses than males at 15, 30, and 45 min after ACTH, but the sexes did not differ in response to the other stimuli. For individual rats, the maximum response to ACTH was slightly correlated with the maximum response to novelty stimulation, nicotine, and saline but was not correlated with the response to LPS.

A rodent model for hemoglobin switching utilizing high performance liquid chromatography.

It has long been recognized that a treatment for beta hemoglobin chain anomalies could result if a way to reverse the Hb F to Hb A switch in humans were found. Studies of hemoglobin switching have been hampered by the fact that small animals normally used in the laboratory do not have a true Hb F. However, several small animal models which take advantage of a switch in minor beta chain proportions in certain strains of inbred mice and rats have been proposed and used. The use of these models has suffered from what, until now, could be considered technically demanding, time-consuming methodologies. In this study we report an effective, rapid and technically streamlined model of hemoglobin switching utilizing Fisher 344 rats and high performance liquid chromatography with a weakly cationic column.

Histopathologic observations in weanling B6C3F1 mice and F344/N rats and their adult parental strains.

Weanling Fischer 344/N (F344) rats and the first filial hybrid of C57BL/6 x C3H (B6C3F1) mice and retired breeders from the parental stocks of these strains were monitored over a 5-yr-period by examining the histopathology of selected organs and comparing those results to viral and mycoplasmal serology and the intestinal tract bacterial flora of each animal on an individual basis. Serology gave no evidence of viral infection, but Mycoplasma arthriditis antibodies were detected. Reactivity of serum of adult C57BL/6 female mice with control cells or media (tissue culture, TC) was seen in a significant number of mice. TC reactivity correlated positively with lymphoid perivascular infiltrates, predominantly of the lungs, suggesting an allergic response in development of the lesions. Other lesions of note consisted of Harderian gland inflammation of rats, focal necrotizing lesions of the liver of both species, and thickening of the pleura and adjacent pulmonary interstitium of weanling rats. Embolization of bacteria from the gastrointestinal tract to the liver was considered a possible cause of the liver necrosis in both species. Although lesions of the lung and Harderian gland of the rats are similar to those caused by known viral agents, the cause of the latter could not be determined as these animals were negative for viral antibodies and the former was considered to be related to incomplete pulmonary development in the young rat. Features differentiating the lesions observed in animals of this survey from those caused by viral infection are discussed.

Age-related changes in the haematology of female F344 rats.

As little comprehensive baseline data are available on age-related haematological changes in genetically-defined rat strains, the haematology of female F344 rats is described in animals sampled at 2, 4, 8, 20, 66 and 121 weeks of age. Values for Hb, RBC and PCV increased from 2 weeks of age to reach adult levels at 8 weeks, whereas MCV, MCH and reticulocyte counts were high initially but decreased to reach the adult range at 8 weeks. Between 66 and 121 weeks, reticulocyte counts were significantly increased and values for MCHC significantly decreased. Lymphocytes were the predominant white cell type in each age group. The absolute numbers of neutrophils and lymphocytes showed slight variations between 2 and 66 weeks and both cell types increased significantly between 66 and 121 weeks. Platelet counts showed no overall age-related trends. Fibrinogen values increased from 2 weeks of age to reach the adult level at 8 weeks. One animal of the 14 sampled at 121 weeks showed changes in the blood, liver and spleen consistent with a diagnosis of lymphoid leukaemia.

Endogenous digoxin-like material in normotensive and hypertensive strains of rats.

We measured the concentration of endogenous digoxin-like materials (EDLM) in the serum of spontaneously hypertensive rats (SHR) and three normotensive rat strains at four stages during growth using a sensitive RIA. In the SHR, there was a significant peak in the EDLM level between 0.057-0.087 ngE/mL at 6 to 8 weeks of age, shortly after the onset of hypertension. The EDLM concentration returned to normal levels by 20 weeks of age. Sprague-Dawley and Wistar-Kyoto rats had EDLM levels below 0.050 ngE/mL at all time points studied. In contrast, Fischer 344 rats displayed persistently elevated serum EDLM concentrations that exceeded 0.124 ngE/mL from 3 to 20 weeks of life. We conclude that (1) there are significant interstrain differences in serum EDLM levels in rats; and (2) the SHR has a unique peak in serum EDLM levels at 6 to 8 weeks of age, indicating a possible role for the substance in the inception of hypertension.

Thyroxine (T4) and triiodothyronine (T3) levels in the Fischer 344 inbred rat.

Plasma levels of T3 and T4 were measured in male and female Fischer 344 (F344/Cr1) inbred rats aged 10, 17, 30, 56, 80 and 106 weeks in order to document the hormone profile of this strain of rat. In general, levels of T4 were higher in males than in females. Levels of T3 were lower in females for the first 17 weeks, similar for rats of age 30 weeks, and thereafter higher in the females than the males. T4 concentration decline was age-associated in both sexes. T3 levels declined in the males only, but remained relatively constant in the females throughout their lifespan. Generally, T4/T3 ratios declined throughout the lifespan of the Fischer rat.

Effect of sampling site and collection method on variations in baseline clinical pathology parameters in Fischer-344 rats. II. Clinical hematology.

An analysis of the influence of blood-sampling site and collection method on peripheral hematology parameters was conducted in age-matched male Fischer-344 rats. Sites examined for blood collection were the right ventricle, abdominal aorta, abdominal vena cava, retroorbital plexus, and tail. Collection methods used included syringe (10 ml), Vacutainer(s) (3 ml or 3 ml X 2), and capillary tube. Blood collected from the tail exhibited leukocyte counts approximately twice those of samples samples from other sites. Blood collected from the retroorbital plexus and tail exhibited significant variations in white blood cell count, red blood cell count, hemoglobin, and hematocrit, and differences in leukocyte differential counts of lymphocytes and neutrophils when compared with other sites. Blood collected from the abdominal aorta and in a second Vacutainer from the right ventricle exhibited lower erythrocyte, leukocyte, and platelet counts than that collected from other sites with the exception of the platelet count from tail blood which was lower than that from all other sites. Although parameter values vary with sample site selection, those obtained from right ventricle blood were the least variable and the most consistent when compared with all other methods.

A comparison of the response of woodchucks and rats to variations in dietary lipotrope and lipid content.

A soy protein-based experimental diet for woodchucks (Marmota monax) is described. The diet supported growth of juvenile woodchucks for 12 wk. With this diet, the effects on both woodchucks and rats of increasing dietary corn oil from 5 to 15% and of deleting supplemental lipotropic factors (choline, methionine, folic acid and vitamin B-12) were studied in a 2 X 2 factorial experiment. Both increased lipid and lipotrope deletion resulted in decreased growth in rats, but only increased lipid caused growth depression in woodchucks. Lipotrope depletion resulted in elevated serum markers of hepatic injury and hepatic lipid accumulation in rats but not in woodchucks. Hematological changes induced by the low lipotrope diets included decreased packed cell volume, total hemoglobin and mean corpuscular volume (MCV) in rats but increased MCV in woodchucks. The woodchuck appears to be more resistant than the rat to induction of hepatic injury by lipotrope deficiency.

A rapid method for repeated collection of blood from the tail vein of rats.

A method was developed for obtaining serial blood samples from the tail vein of rats. Seventy microliters of blood were collected at the rate of approximately one sample per minute for the quantitative determination of whole blood clearance of radiolabeled compounds. The technique was rapid, reproducible, and kept stress to the test animals at a minimum.

Measurement of plasma LH concentrations in aged male rodents by a radioimmunoassay and a radioreceptor assay.

Circulating plasma concentrations of LH from young mature (3-4 months old), middle-aged (15-18 months old) and aged (31-32 months old) male C57BL/6 mice, Syrian hamsters (3-4, 19-20 and 24-25 months old), Fischer 344 rats (3-4, 18-19 and 28-29 months old), Chinese hamsters (3-4, 19-20 and 29-30 months old) and Mongolian gerbils (3-4 and 19-22 months old) were analysed using a radioimmunoassay (RIA) and a radioreceptor assay (RRA). Male rats exhibited the greatest changes with advancing age: the oldest rats had an almost undetectable quantity of plasma LH, as measured by both assays. In contrast, the oldest male Syrian hamsters had significantly higher levels of LH than did younger animals. A significant decrease occurred in the amounts of LH detectable by RRA in middle-aged Chinese hamsters which was not evident with the RIA. There were no statistically significant differences in LH levels of C57BL/6 mice and gerbils with increasing age. The mean RRA:RIA ratios indicated that age-related differences in LH concentrations resulted from physiological changes in the secretion or the metabolic clearance of LH and not from changes in the biological potency of LH.

Hematologic and serum chemical characteristics of mononuclear leukemia in Fischer 344 rats.

Hematologic, serum chemical, and histopathologic studies were performed on 17 aged Fischer 344 rats with mononuclear leukemia. Twelve of the rats had leukemic hemograms, while five had nonleukemic or marginally abnormal differential leukocyte counts. Hematologic findings revealed that all rats were profoundly anemic. Serum chemistry studies confirmed the occurrence of icterus observed clinically, a finding consistent with hemolytic anemia. Alanine aminotransferase and serum alkaline phosphatase values were elevated.

Concomitant changes of ACTH, beta-endorphin and N-terminal portion of pro-opiomelanocortin in rats.

Radioimmunoassay developed to measure N-terminal peptide of pro-opiomelanocortin isolated from porcine pituitaries was used to measure changes in the concentration of immunoreactive material in rat plasma. The N-terminal peptide immunoreactive material decreased in plasma after hypophysectomy of both female and male rats below the level of detectability and substantially increased after adrenalectomy as compared to normal control rats. The same changes were observed when beta-endorphin and ACTH like immunoreactive material was measured. The primary culture of rat anterior pituitary cells released ACTH and N-terminal peptide-like immunoreactive material into the incubation medium. The results seem to indicate that the N-terminal immunoreactive material is a secretory product produced by the pituitary gland.