Quantitative relationship between plasma potassium levels and QT interval in beagle dogs.

The aim of the current study was to establish the quantitative relationship between plasma potassium concentrations and the QT interval of the electrocardiogram in dogs. Furosemide, a potent diuretic, was given at increasing doses (5-60 mg/kg) to five male and five female beagle dogs. Electrocardiogram (ECG) was recorded three times each day, simultaneous to blood sampling for measurement of plasma potassium. Furosemide treatment produced a clear hypokalaemia, which was associated with an increase in QT and corrected QT intervals (QTc) duration. On average, the slopes of the negative linear correlation between potassium plasma levels and QT or QTc were steeper in females than in males. These results show that a decrease in potassium plasma level may explain a concomitant increase in QT duration in a toxicity study in dogs, in particular if potassium values are decreased below 3.3 mmol/L. Correction of QT interval for K+ plasma level has, therefore, been established separately for males and females. A global formula correcting QT for K+ and heart rate simultaneously was established. Hypokalaemia was also associated with changes in the morphology of the T wave recorded in CV5RL, in particular, with a flattening and/or a notching of the wave (appearance of a second peak), biphasic aspect or inversion of polarity. These changes are probably related to an increased heterogeneity of repolarization between different populations of cardiomyocytes. In conclusion, hypokalaemia is quantitatively associated with an increase in QT and QTc duration in dogs. The relationship is apparently stronger for females than for males. A formula may be used to correct QT for potassium plasma level.

Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals.

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.

Time course toxicogenomic profiles in CD-1 mice after nontoxic and nonlethal hepatotoxic paracetamol administration.

Adverse drug reactions are a major clinical problem. Drug-induced hepatotoxicity constitutes a large percentage of these reactions. A thorough understanding of the genetic events, specifically, the early "decision-making" processes underlying biological changes caused by drugs and metabolites, is required. To assist in the understanding of these events, we have employed the model hepatotoxin, paracetamol (APAP), and GeneChip technology to investigate global genetic events seen after nontoxic and toxic doses in the mouse. Mice were dosed [vehicle, nontoxic APAP (1 mmol/kg), and toxic APAP (3.5 mmol/kg)], and individual hepatic RNA samples were hybridized to separate chips to determine interanimal variation. Statistical analysis detected 175 CD-1 mouse genes that were significantly regulated (P < 4.1 x 10(-6)), and nonsignificant genes were discarded. For clarity, the significantly regulated genes were then binned into categories according to their major function-antioxidant, glutathione, metabolism, transcription, immune, and apoptosis. There was no hepatic stress observed after dosing 1 mmol/kg APAP, when measured by serum alanine aminotransferase levels. Hepatic toxicity was observed at both 4 and 24 h after a 3.5 mmol/kg dose of APAP. Time course expression profiles for selected genes have been created. These results demonstrate that most active gene expression occurs around 4 h after a toxic dose of APAP. Down-regulation of these genes is observed over 24 h, coinciding with the development of overt toxicity. These data provide a deeper understanding of the in vivo time course of physiological responses of the liver to chemical stress and provide a logical step forward for the investigation of new chemical entities demonstrated positive in chemically reactive metabolite screens. The complete data set can be viewed at http://www.ebi.ac.uk/arrayexpress/. The accession number is E-MEXP-82.

Harmonization of animal clinical pathology testing in toxicity and safety studies. The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing.

Ten scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in regulated toxicity and safety studies. For repeated-dose studies in rodent species, clinical pathology testing is necessary at study termination. Interim study testing may not be necessary in long-duration studies provided that it has been done in short-duration studies using dose levels not substantially lower than those used in the long-duration studies. For repeated-dose studies in nonrodent species, clinical pathology testing is recommended at study termination and at least once at an earlier interval. For studies of 2 to 6 weeks in duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals. If a study contains recovery groups, clinical pathology testing at study termination is recommended. The core hematology tests recommended are total leukocyte (white blood cell) count, absolute differential leukocyte count, erythrocyte (red blood cell) count, evaluation of red blood cell morphology, platelet (thrombocyte) count, hemoglobin concentration, hematocrit (or packed cell volume), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In the absence of automated reticulocyte counting capabilities, blood smears from each animal should be prepared for reticulocyte counts. Bone marrow cytology slides should be prepared from each animal at termination. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis. The core clinical chemistry tests recommended are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. For hepatocellular evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, glutamate dehydrogenase, or total bile acids. For hepatobiliary evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alkaline phosphatase, gamma glutamyltransferase, 5' -nucleotidase, total bilirubin, or total bile acids. Urinalysis should be conducted at least once during a study. For routine urinalysis, an overnight collection (approximately 16 hr) is recommended. It is recommended that the core tests should include an assessment of urine appearance (color and turbidity), volume, specific gravity or osmolality, pH, and either the quantitative or semiquantitative determination of total protein and glucose. For carcinogenicity studies, only blood smears should be made from unscheduled sacrifices (decedents) and at study termination to aid in the identification and differentiation of hematopoietic neoplasia.

Evaluation of the Clinitek 200 urinary test-strip reader in the analysis of dog and rat urines in pre-clinical toxicology studies.

The performance of a clinical urinary test-strip reader Clinitek 200 was evaluated for dog and rat urines, in the context of pre-clinical toxicology studies. No major discrepancies were found between data generated by visual estimation or automatic measurement. Analysis of spiked samples showed good agreement between actual concentrations and Clinitek 200 responses for ketone bodies and glucose although a lack of sensitivity was found for the latter. Results for proteins showed over- or underestimation in dog and rat urines respectively at low concentrations, and overestimation at high concentrations in both species. Reproducibility of responses was excellent for ketone bodies, glucose and proteins but was weaker for haemoglobin and bilirubin. High bilirubin concentrations were found to interfere with the haemoglobin reaction. The pH measurements were found to be accurate only around pH 7. Specific gravity measurements were unreliable. Overall, the Clinitek 200 as a screening tool proved sufficiently reliable in the measurement of all parameters tested, with the exception of specific gravity.

An investigation of urinary creatine excretion as a potential marker for testicular damage.

1. This study assessed urinary creatine excretion as a marker for testicular atrophy. 2. Male rats received a single i.p. dose of 2-methoxyethanol at 0, 250 or 750 mg kg-1 and were sacrificed 2 d later. Urinary creatine and creatinine excretion were measured on days 0, 1 and 2. 3. Decreased testicular weights and histopathological assessment revealed dose-related testicular damage. 4. On day 1, at both doses of 2-methoxyethanol, urinary creatine levels increased and creatinine levels decreased, resulting in a dose-related increase in the creatine/creatinine ratio. On day 2, the creatine/creatinine ratio was elevated relative to controls, but was less marked than on day 1. 5. The study confirmed that creatine excretion is a potential marker for acute testicular damage.

Fat, frail and dying young: survival, body weight and pathology of the Charles River Sprague-Dawley-derived rat prior to and since the introduction of the VAFR variant in 1988.

Trends in survival and body weight were evaluated in 2140 control Sprague-Dawley-derived [Crl: COBS-CD(SD)BR and Crl: COBS-VAF CD(SD)BR] rats used for 24-month rat carcinogenicity studies between 1979 and 1991. Body weight and survival were remarkably stable in the CD-COBS rats used during 1979-1987: at 24 months, the mean survival in males was 68 +/- 5%, and 60 +/- 5% in females. With the CD-COBS-VAF rat, a variant of the CD-COBS strain used between 1988 and 1991, the survival at 24 months dropped to 41 +/- 3% in males, and 44 +/- 7% in females compared to the CD-COBS. The CD-COBS-VAF rat had a significantly reduced life span (P < 0.001 at 24 months), a significant increase in mean body weight (males at 6 months: 672 +/- 24 g vs. 536 +/- 6 g; females: 359 +/- 7 g vs 308 +/- 3 g; P < 0.001) and food consumption (males at 6 months: 31.3 +/- 3.3 vs. 25.4 +/- 2.1 g d-1; females: 22.0 +/- 2.7 g v. 20.3 +/- 2.0 g d-1; P < 0.001). CD-COBS-VAF rats which failed to survive up to study termination had individual body weights at 3, 6 and 12 months which were significantly higher (P < 0.001) than those which survived until 24 months. Our historical data base of control rats (CD-COBS and CD-COBS-VAF) in carcinogenicity studies revealed a significant (males: P < 0.001); females: P < 0.01) and inverse linear relation between mean 3-month body weight and 24-month survival. When compared to CD-COBS animals, CD-COBS-VAF rats showed an increase in the incidence of pituitary tumours in males, mammary fibroadenomas in females, an increase in the incidence of severity of glomerulonephrosis, and a greater incidence of animals which died without any obvious pathology. It is concluded that, in our Sprague-Dawley substrains, both the individual and the group mean body weights in early adult life appear predictive for the individual and group life expectancy. The decrease in longevity in the CD-COBS-VAF rat is principally due to disease and degeneration processes associated with fast growth and high body weight.

Variations of sodium, potassium, and chloride plasma levels in the rat with age and sex.

Sodium, potassium, and chloride plasma levels were measured in 294 male and 286 female Sprague Dawley rats [Crl:COBS CD(SD)BR]. The rats were distributed between four groups according to age (65-125 days, 235-275 days, 353-482 days, and 665-775 days). The levels of all three ions were higher in males than in females: about 1% higher for sodium and chloride and about 7% higher for potassium. Potassium and chloride values decreased with increasing age in both sexes; potassium decreased 12% and chloride decreased 6%. Distributions were not perfectly Gaussian but the departures from normality were slight. It was concluded, therefore, that determinations based on parametric statistical tests on the data are unlikely to be seriously biased by the distribution.