Correlation of serum cardiac troponin I and acute phase protein concentrations with clinical staging in dogs with degenerative mitral valve disease.

BACKGROUND: Cardiac troponin I (cTnI) correlates with severity of myocardial injury. Nonspecific inflammation in congestive heart failure (CHF) could be assessed by C-reactive protein (CRP), haptoglobin (Hp), and ceruloplasmin (Cp) measurements. OBJECTIVES: The aim of the study was to determine whether serum cTnI, CRP, Hp, and Cp concentrations differ among various stages of mitral valve disease (MVD) in dogs. MATERIALS AND METHODS: Dogs with MVD were allocated to 3 groups (I - asymptomatic; II - mild to moderate CHF; III advanced CHF) according to the scheme of the International Small Animal Cardiac Healthy Council (ISACHC). Concentrations of cTnI, CRP, Cp, and Hp were measured in all dogs upon admission, and cTnI and CRP were measured bimonthly during a 4-month follow-up period. RESULTS: In total 46 dogs with MVD were enrolled for the cross-sectional part (21 Group I, 11 Group II, 14 Group III), and 35 dogs were included in the longitudinal study. Initial mean Cp concentrations were similar among all groups. There was a statistically significant difference in Hp and CRP concentrations between group I (n = 21, P = .019) and III (n = 14, P < .001). There was a statistically significant decrease in CRP (P = .033) and cTnI (P = .009) concentrations over the longitudinal study (all groups). CRP concentrations were significantly higher in group I than III (P = .004). During the 6-month monitoring period of 35 dogs, there was a statistically significant positive correlation between cTnI and CRP (P < .001). CONCLUSION: Differences in CRP concentrations between clinical stages of MVD suggest a clinically and therapeutically relevant inflammatory component.

Serial analysis of serum cardiac troponin I changes and correlation with clinical findings in 46 dogs with mitral valve disease.

BACKGROUND: Cardiac troponin I (cTnI) is a biomarker correlated with the severity of myocardial injury. It is hypothesized that serial assessment of cTnI could provide information about the disease progression in chronic heart failure. OBJECTIVE: The purpose of the study was to correlate serial serum cTnI concentrations with clinical scoring and select diagnostic imaging findings in dogs managed for mitral valve degeneration (MVD) for a period of 6 months. METHODS: Client-owned dogs with MVD were prospectively recruited for the study. The dogs were allocated into 3 groups (I, II, III) according to the severity of their clinical signs based on the classification suggested by the International Small Animal Cardiac Health Council. During the 6-month study period, serum specimens for biochemical testing were obtained biweekly, clinical progression and response to treatment were also evaluated biweekly, and radiographic reevaluation was performed every 2 months. RESULTS: A total of 46 dogs were evaluated. There was a marked decrease in cTnI values during the first 2 weeks after initial diagnosis, more pronounced in group III, and corresponding to the initiation of therapy and clinical stabilization of animals. Serum cTnI was significantly different (P < .05) among the 3 dog groups throughout the study period. The interaction between Group and Period was significant in group III, indicating an upward cTnI trend evident in severely affected animals. A positive correlation was demonstrated in all groups between serum cTnI and clinical scoring. CONCLUSION: Long-term management of MVD in dogs could benefit from the serial measurement of cTnI.

Spontaneous cardiomyopathy in young Sprague-Dawley rats: evaluation of biological and environmental variability.

Cardiovascular safety signals in nonclinical studies remain among the main reasons for drug attrition during pharmaceutical research and development. Drug-induced changes can be functional and/or associated with morphological alterations in the normal heart histology. It is therefore crucial to understand the normal variations in histology to discriminate test article-related changes from background lesions. Rodent progressive cardiomyopathy is probably the most commonly encountered change in control animals of nonclinical toxicity studies. A multisite study mimicking standard short-term toxicity studies using young male Sprague-Dawley rats was performed to better characterize this finding. Using an enhanced sectioning method for this research study, it was observed that the incidence of background cardiomyopathy was 100%. The vast majority of the microscopic findings were inflammatory in nature, with associated necrotic changes (defined as necrosis/inflammatory cell infiltrate) and these changes were mainly located in the myocardium of the mid region of the ventricles (the left side being predominantly affected). The monitored environmental factors in this study (multiple facilities, study duration, handling) did not have an effect on the incidence or severity of the spontaneous cardiomyopathy. In addition, cardiac-specific serum troponin levels were measured and were within the published control range.

Comparison of cardiac troponin I and T, including the evaluation of an ultrasensitive assay, as indicators of doxorubicin-induced cardiotoxicity.

Cardiac troponin (cTn) has been utilized to assess acute myocardial injury, but the cTn response in active/ongoing chronic injury is not well documented. The purpose of this study was to characterize the cardiac troponin I (cTnI), cardiac troponin T (cTnT), high-sensitivity cTnI, hematology, and clinical chemistry responses in rats treated with doxorubicin. Rats treated with 1, 2, or 3 mg/kg/week (wk) of doxorubicin for 2, 4, or 6 wks were sacrificed after 0, 2, or 4 wks of recovery and compared to untreated controls and animals treated with doxorubicin/dexrazoxane (50 mg/kg/wk) or etoposide (1 and 3 mg/kg/wk). The incidence and mean magnitude of cTn response increased with increasing dose and/or duration of doxorubicin treatment. Conversely, dexrazoxane/doxorubicin was partially protective for cardiotoxicity, and minimal cardiotoxicity occurred with etoposide treatment. Both cTnI and cTnT effectively identified doxorubicin-induced injury as indicated by vacuolation of cardiomyocytes of the atria/ventricles. The association between the cTn responses and histological changes was greater at the higher total exposures, but the magnitude of cTn response did not match closely with histologic grade. The high-sensitivity cTnI assay was also effective in identifying cardiac injury. Alterations occurred in the hematology and clinical chemistry parameters and reflected both dose and duration of doxorubicin treatment.

Correlation of histopathology, urinary biomarkers, and gene expression responses following hexachloro-1:3-butadiene-induced acute nephrotoxicity in male Hanover Wistar rats: a 28-day time course study.

Hexachloro-1:3-butadiene (HCBD) causes segment-specific injury to the proximal renal tubule. A time course study of traditional and more recently proposed urinary biomarkers was performed in male Hanover Wistar rats receiving a single intraperitoneal (ip) injection of 45 mg/kg HCBD. Animals were killed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 28 postdosing and the temporal response of renal biomarkers was characterized using kidney histopathology, urinary and serum biochemistry, and gene expression. Histopathologic evidence of tubular degeneration was seen from day 1 until day 3 postdosing and correlated with increased urinary levels of α-glutathione S-transferase (α-GST), albumin, glucose, and kidney injury molecule-1 (KIM-1), and increased gene expression of KIM-1, NAD(P)H dehydrogenase, quinone 1, and heme oxygenase (decycling) 1. Histopathologic evidence of tubular regeneration was seen from day 2 postdosing and correlated with raised levels of urinary KIM-1 and osteopontin and increased gene expression of KIM-1 and annexin A7. Traditional renal biomarkers generally demonstrated low sensitivity. It is concluded that in rat proximal tubular injury, measurement of a range of renal biomarkers, in conjunction with gene expression analysis, provides an understanding of the extent of degenerative changes induced in the kidney and the process of regeneration.

Nephrotoxicity of hexachloro-1:3-butadiene in the male Hanover Wistar rat; correlation of minimal histopathological changes with biomarkers of renal injury.

Hexachloro-1:3-butadiene (HCBD) causes damage specifically to the renal proximal tubule in rats. In the present study, injury to the nephron of male Hanover Wistar rats was characterized at 24 h following dosing with HCBD in the range 5-90 mg kg⁻¹ to determine the most sensitive biomarkers of damage, that is, the biomarkers demonstrating significant changes at the lowest dose of HCBD, using a range of measurements in serum and urine, renal histopathology, and renal and hepatic gene expression. Histologically, kidney degeneration was noted at doses as low as 10 mg kg⁻¹ HCBD. Significant changes in the hepatic and renal gene expression categories of xenobiotic metabolism and oxidative stress were observed at 5 mg kg⁻¹ HCBD, and in the kidney alone, evidence of inflammation at 90 mg kg⁻¹ HCBD. Increases in the urinary excretion of α-glutathione S-transferase (α-GST) and kidney injury molecule-1 (KIM-1) were seen at 10 mg kg⁻¹ HCBD, and increases in urinary excretion of albumin and total protein were evident at 15 mg kg⁻¹ HCBD. The most sensitive, noninvasive biomarkers of HCBD-induced renal toxicity in Hanover Wistar rats were urinary α-GST and KIM-1. Urinary albumin measurement is also recommended as, although it is not the most sensitive biomarker, together with α-GST, albumin showed the largest relative increase of all the biomarkers investigated, and the protein is easily measured.

Urinary biomarkers in hexachloro-1:3-butadiene-induced acute kidney injury in the female Hanover Wistar rat; correlation of α-glutathione S-transferase, albumin and kidney injury molecule-1 with histopathology and gene expression.

Hexachloro-1:3-butadiene (HCBD) causes kidney injury specific to the pars recta of the proximal tubule. In the present studies, injury to the nephron was characterized at 24 h following a single dose of HCBD, using a range of quantitative urinary measurements, renal histopathology and gene expression. Multiplexed renal biomarker measurements were performed using both the Meso Scale Discovery (MSD) and Rules Based Medicine platforms. In a second study, rats were treated with a single nephrotoxic dose of HCBD and the time course release of a range of traditional and newer urinary biomarkers was followed over a 25 day period. Urinary albumin (a marker of both proximal tubular function and glomerular integrity) and α-glutathione S-transferase (α-GST, a proximal tubular cell marker of cytoplasmic leakage) showed the largest fold change at 24 h (day 1) after dosing. Most other markers measured on either the MSD or RBM platforms peaked on day 1 or 2 post-dosing, whereas levels of kidney injury molecule-1 (KIM-1), a marker of tubular regeneration, peaked on day 3/4. Therefore, in rat proximal tubular nephrotoxicity, the measurement of urinary albumin, α-GST and KIM-1 is recommended as they potentially provide useful information about the function, degree of damage and repair of the proximal tubule. Gene expression data provided useful confirmatory information regarding exposure of the kidney and liver to HCBD, and the response of these tissues to HCBD in terms of metabolism, oxidative stress, inflammation, and regeneration and repair.

Haemotoxicity of busulphan, doxorubicin, cisplatin and cyclophosphamide in the female BALB/c mouse using a brief regimen of drug administration.

Many anticancer drugs are myelotoxic and cause bone marrow depression; however, generally, the marrow/blood returns to normal after treatment. Nevertheless, after the administration of some anti-neoplastic agents (e.g. busulphan, BU) under conditions as yet undefined, the marrow may begin a return towards normal, but normality may not be achieved, and late-stage/residual marrow injury may be evident. The present studies were conducted to develop a short-term mouse model (a 'screen') to identify late-stage/residual marrow injury using a brief regimen of drug administration. Female BALB/c mice were treated with BU, doxorubicin (DOX), cisplatin (CISPLAT) or cyclophosphamide (CYCLOPHOS) on days 1, 3 and 5. In 'preliminary studies', a maximum tolerated dose (MTD) for each drug was determined for use in 'main studies'. In main studies, mice were treated with vehicle (control), low and high (the MTD) dose levels of each agent. Necropsies were performed, and blood parameters and femoral/humeral nucleated marrow cell counts (FNCC/HNCC) were assessed on six occasions (from days 1 to 60/61 post-dosing). Late-stage/residual changes were apparent in BU-treated mice at day 61 post-dosing: RBC, Hb and haematocrit were reduced, mean cell volume/mean cell haemoglobin were increased and platelet and FNCC counts were decreased. Mice given DOX, CISPLAT and CYCLOPHOS, in general, showed no clear late-stage/residual effects (day 60/61). It was concluded that a brief regimen of drug administration, at an MTD, with assessment at day 60/61 post-dosing was a suitable short-term method/screen in the mouse for detecting late-stage/residual marrow injury for BU, a drug shown to exhibit these effects in man.

Time course characterization of serum cardiac troponins, heart fatty acid-binding protein, and morphologic findings with isoproterenol-induced myocardial injury in the rat.

We investigated the kinetics of circulating biomarker elevation, specifically correlated with morphology in acute myocardial injury. Male Hanover Wistar rats underwent biomarker and morphologic cardiac evaluation at 0.5 to seventy-two hours after a single subcutaneous isoproterenol administration (100 or 4000 microg/kg). Dose-dependent elevations of serum cardiac troponins I and T (cTnI, cTnT), and heart fatty acid-binding protein (H-FABP) occurred from 0.5 hour, peaked at two to three hours, and declined to baseline by twelve hours (H-FABP) or forty-eight to seventy-two hours (Serum cTns). They were more sensitive in detecting cardiomyocyte damage than other serum biomarkers. The Access 2 platform, an automated chemiluminescence analyzer (Beckman Coulter), showed the greatest cTnI fold-changes and low range sensitivity. Myocardial injury was detected morphologically from 0.5 hour, correlating well with loss of cTnI immunoreactivity and serum biomarker elevation at early time points. Ultrastructurally, there was no evidence of cardiomyocyte death at 0.5 hour. After three hours, a clear temporal disconnect occurred: lesion scores increased with declining cTnI, cTnT, and H-FABP values. Serum cTns are sensitive and specific markers for detecting acute/active cardiomyocyte injury in this rat model. Heart fatty acid-binding protein is a good early marker but is less sensitive and nonspecific. Release of these biomarkers begins early in myocardial injury, prior to necrosis. Assessment of cTn merits increased consideration for routine screening of acute/ongoing cardiomyocyte injury in rat toxicity studies.

Cardiac troponin I in isoproterenol-induced cardiac injury in the Hanover Wistar rat: studies on low dose levels and routes of administration.

The current studies demonstrate the effect of low-dose intraperitoneal (IP) administration of isoprotenerol (ISO) and subcutaneous (SC) versus IP routes of administration of ISO on serum cardiac troponin I (cTnI) levels in female Hanover Wistar rats, providing additional evidence to support acceptance of cTnI as a cardiac biomarker. At 2 hr postdosing with 0-500 microg/kg ISO, mean serum cTnI levels were increased in a dose-related fashion at > or =10 microg/kg with no evidence of cardiac pathology. At 24 h, cTnI concentrations were generally at control levels, but histologic cardiomyocyte injury was evident in a proportion of the animals given > or =10 microg/kg. In a second experiment, rats given SC ISO at 5,000 microg/kg and necropsied at 0, 1, 2, and 4 hr postdosing had higher levels of serum cTnI than animals given the same dose IP.

Dose response and time course studies on superoxide dismutase as a urinary biomarker of carbon tetrachloride-induced hepatic injury in the Hanover Wistar rat.

Previous studies have shown that the enzyme copper/zinc superoxide dismutase (SOD-1) is increased in the urine of rats with carbon tetrachloride (CCl(4))-induced hepatotoxicity. The present experiments aimed to investigate further the usefulness of urinary SOD-1 as a non-invasive biomarker of liver injury. Two investigations were carried out, a dose response study and a time course study. In the dose response study, rats were given a single dose of CCl(4) at 0 (control), 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.80 ml/kg and urine samples collected from 12 to 36 h postdosing. In the time course study, rats were dosed at 0.80 ml/kg CCl(4) and urine sampled at 4, 12, 24 and 36 h postdosing. In both studies, the presence of SOD-1 in the urine was confirmed by Western blotting with an SOD-1 antibody. In the dose response study, serum SOD activity was elevated in all CCl(4)-treated animals and urinary SOD-1 activity was increased 2.2 times at the lowest dose (0.10 ml/kg) and 60.4 times at the highest CCl(4) dose level (0.80 ml/kg). In the time course study, urinary SOD-1 was first detected in samples collected from 4 to 12 h postdosing. We conclude that urinary SOD-1 has potential as a sensitive non-invasive biomarker of CCl(4)-induced hepatocellular injury.

A translational approach to detecting drug-induced cardiac injury with cardiac troponins: consensus and recommendations from the Cardiac Troponins Biomarker Working Group of the Health and Environmental Sciences Institute.

Cardiac troponins (cTns) are established biomarkers of ischemic heart disease in humans. However, their value as biomarkers of cardiac injury from causes other than ischemic heart disease is now being explored, particularly in drug development. In a workshop sponsored by the Cardiac Troponin Biomarker Working Group of the Health and Environmental Sciences Institute, preclinical, clinical, and regulatory scientists discussed the application of cTns in their respective environments, issues in translating the preclinical application of cTn to clinical studies, and gaps in our understanding of cTn biology and pathobiology. Evidence indicates that cTns are sensitive and specific biomarkers of cardiac injury from varying causes in both animals and humans. Accordingly, monitoring cTns can help ensure patient safety during the clinical evaluation of new drugs. In addition, preclinical characterization of cardiac risk and cTns as biomarkers of that risk can guide relevant clinical application and interpretation. We summarize here the outcomes of the workshop which included consensus statements, recommendations for further research, and a proposal for a cross-disciplinary group of clinical, regulatory, and drug development scientists to collaborate in such research.

Proteomic investigation of urinary markers of carbon-tetrachloride-induced hepatic fibrosis in the Hanover Wistar rat.

Proteomic techniques such as two-dimensional gel electrophoresis (2-DGE) and mass spectrometry have become important tools for the identification of novel biomarkers of toxicity and disease. Ideally, such biomarkers need to be sensitive and organ specific, but, recently, it has become apparent that it would be an additional benefit to be able to measure biomarkers in samples obtained using non-invasive methods. The present study is concerned with the identification of novel urinary markers of hepatic fibrosis. In a carbon-tetrachloride-induced liver fibrosis rat model, analysis of urine by 2-DGE revealed an increase in the concentration of a number of proteins in animals with hepatic fibrosis. Using in-gel trypsin digest and nano-scale liquid chromatography combined with electrospray ionisation tandem mass spectrometry, protein spots were identified as copper/zinc superoxide dismutase, D: -dopachrome tautomerase, beta-2-microglobulin and neutrophil gelatinase associated lipocalin. These proteins are known to have important roles in the inflammatory response.

Analytical characteristics of commercial cardiac troponin I and T immunoassays in serum from rats, dogs, and monkeys with induced acute myocardial injury.

BACKGROUND: Information is needed regarding analytical characteristics of cardiac troponin (cTn) assays used in preclinical studies. METHODS: We measured cTnI and cTnT in serum from normal animals and animals with induced myocardial injury [Sprague-Dawley (SD) and Wistar rats, beagle dogs, and rhesus (Rh) and cynomolgus (Cy) monkeys]. We evaluated the following assays: for cTnI, Abbott Architect, Bayer Centaur (first and second generation), Beckman Access, DPC Immulite, Dade Dimension, Ortho Vitros ES, Tosoh AIA, and species-specific enzyme immunoassays; for cTnT, Roche Elecsys. RESULTS: We found different species-specific responses for the troponin assays evaluated. Abbott, Bayer Ultra, Beckman, and Dade assays gave good responses across all species. In rats, weak responses were observed with DPC and Ortho, and no measurable response with Tosoh. In dogs, weak responses were observed with Tosoh cTnI, Roche cTnT, and species-specific cTnI. In cynomolgus monkeys, weak responses were observed with species-specific cTnI and Roche cTnT. Assay imprecision was < or = 20% at 3 or more examined cTn concentrations for Beckman (rat, dog, monkey), Dade (rat, dog, monkey), Abbott (rat, dog, monkey), Bayer first generation (dog), Bayer Ultra (rat, dog, monkey), Roche (monkey), DPC (dog, monkey), Ortho (dog, monkey), and Tosoh (dog, monkey) assays, whereas imprecision was < or = 20% at 2 or fewer concentrations for the Bayer first generation (rat, monkey), Roche cTnT (rat, dog), and DPC (rat) assays. CONCLUSIONS: Not all cTn assays are suitable for monitoring cTn in each animal species or strain. Individual assay characterization by animal species is needed to prevent misinterpretation of myocardial injury-based cardiac troponin findings.

Comprehensive characterization of serum clinical chemistry parameters and the identification of urinary superoxide dismutase in a carbon tetrachloride-induced model of hepatic fibrosis in the female Hanover Wistar rat.

Carbon tetrachloride (CCl(4)) was used to induce liver fibrosis in the rat. Using this model, we have identified changes in serum and urinary clinical chemistry parameters, and characterized histopathological lesions in the liver. Two experiments were conducted. In Experiment 1, rats were dosed at six levels of CCl(4) (0.06-0.36 ml/kg) twice weekly for 6 weeks, followed by a 6-week non-dosing recovery period (week 12). Livers were removed for histology at 6 and 12 weeks and serum parameters analysed. In Experiment 2, rats were given seven dose levels of CCl(4) (0.4-1.0 ml/kg) twice weekly for 6 weeks, followed by a 6-week recovery period (week 12); urine samples were analysed at 3, 6, 9 and 12 weeks using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liver fibrosis was evident at 6 weeks in Experiments 1 and 2, and the activity of serum enzymes (including alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase) was increased. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis (Experiment 2) revealed a protein band at 18.4 kDa in urine from rats treated with CCl(4), not present in control urine, which was identified as copper/zinc superoxide dismutase (Cu/Zn SOD). Western blotting revealed that SOD was increased in urine from rats treated with CCl(4) at 3 and 6 weeks, but not at 9 and 12 weeks. We conclude that Cu/Zn SOD is a urinary marker of hepatic necrosis, but not hepatic fibrosis.

Characterization of troponin responses in isoproterenol-induced cardiac injury in the Hanover Wistar rat.

The investigations aimed to evaluate the usefulness of cardiac troponins as biomarkers of acute myocardial injury in the rat. Serum from female Hanover Wistar rats treated with a single intraperitoneal (IP) injection of isoproterenol (ISO) was assayed for cardiac troponin I (cTnI) (ACS: 180SE, Bayer), cTnI (Immulite 2000, Diagnostic Products Corporation) and cardiac troponin T (cTnT) (Elecsys 2010, Roche). In a time-course study (50.0 mg/kg ISO), serum cTnI (ACS:180SE) and cTnT increased above control levels at 1 hour postdosing, peaking at 2 hours (cTnI, 4.30 microg/L; cTnT, 1.79 microg/L), and declined to baseline by 48 hours, with histologic cardiac lesions first seen at 4 hours postdosing. The Immulite 2000 assay gave minimal cTnI signals, indicating poor immunoreactivity towards rat cTnI. In a dose-response study (0.25 to 20.0 mg/kg ISO), there was a trend for increasing cTnI (ACS:180SE) values with increasing ISO dose levels at 2 hours postdosing. By 24 hours, cTnI levels returned to baseline although chronic cardiac myodegeneration was present. We conclude that serum cTnI and cTnT levels are sensitive and specific biomarkers for detecting ISO induced myocardial injury in the rat. Serum troponin values reflect the development of histopathologic lesions; however peak troponin levels precede maximal lesion severity.

Alpha-glutathione S-transferase in the assessment of hepatotoxicity--its diagnostic utility in comparison with other recognized markers in the Wistar Han rat.

The diagnostic utility of alpha-glutathione S-transferase (alphaGST) in the assessment of acute hepatotoxicity was compared with a range of markers including alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Rats were given a single oral dose of either alpha-naphthylisothiocynate (AN IT), bromobenzene (BrB). or thioacetamide (TAM) at concentrations previously shown to induce marked hepatotoxicity. The progression of each hepatic lesion was monitored by the measurement of a battery of markers, including alphaGST, in plasma collected at time points ranging from 3 h to 7 days after dosing. alphaGST was seen to increase significantly at 24 h (ANIT/BrB) and 3 h (TAM) postdosing, corresponding with histopathological findings. For each compound, when the degree of insult was most severe, fold increases in alphaGST were greater than those seen with ALT and AST, yet lower than those seen with glutamate dehydrogenase (BrB and ANIT). sorbitol dehydrogenase (TAM), or total bilirubin and bile acids (ANIT). Elevations in alphaGST were also detected no earlier than any other marker. AlphaGST in the rat was shown to be a valid marker of hepatotoxicity; however, its measurement offered no additional information in detecting either the time of onset/recovery or the severity of each type of hepatic injury induced.