ABSTRACT
INTRODUCTION: Hemolytic kernicterus, an indirect bilirubin-induced brain dysfunction, is associated with hyper-bilirubinemia in mammalian neonates. In this study, a new model of kernicterus has been developed using intra-peritoneal injections of phenyl hydrazine and subcutaneous injections of sulfisoxazole. These drugs can potentially induce kernicterus in neonatal through changes in hemolysis and hypo-albumin. METHODS: For this purpose, 7-day-old male Wistar rats (n=72; mean weight 11±1g) were used. The animals have been divided into six different groups which received the drugs alone and their combination, and the drugs' solvents and their combination. Biochemical parameters, brain iron and bilirubin, behavioural performance, auditory function and apoptosis were measured using auto-analyser instruments; atomic absorption spectroscopy, Sawasaki, footprint, auditory brainstem response (ABR) and TUNEL test, respectively. RESULT: The drug-injected groups showed a significant reduction in serum haematocrit and an increase in the concentration of brain bilirubin, total and indirect bilirubin as well as TUNEL positive cells in basal ganglia. In addition, the obtained results showed that there was a significant increase in behavioural disturbance and auditory dysfunction in the group injected with the combination of two drugs. CONCLUSION: This kernicterus-induced rat model could perfectly mimic the common conditions of the hyperbilirubinemia in human neonates. This study offers an easy technique to develop more stable models for follow-up studies.
Subject(s)
Bilirubin/metabolism , Disease Models, Animal , Kernicterus/chemically induced , Kernicterus/metabolism , Animals , Animals, Newborn , Evoked Potentials, Auditory, Brain Stem/drug effects , Evoked Potentials, Auditory, Brain Stem/physiology , Kernicterus/pathology , Male , Phenylhydrazines/toxicity , Random Allocation , Rats , Rats, Wistar , Sulfisoxazole/toxicityABSTRACT
Kernicterus is a neurological syndrome associated with indirect bilirubin accumulation and damages to the basal ganglia, cerebellum and brain stem nuclei particularly the cochlear nucleus. To mimic haemolysis in a rat model such that it was similar to what is observed in a preterm human, we injected phenylhydrazine in 7-day-old rats to induce haemolysis and then infused sulfisoxazole into the same rats at day 9 to block bilirubin binding sites in the albumin. We have investigated the effectiveness of human adiposity-derived stem cells as a therapeutic paradigm for perinatal neuronal repair in a kernicterus animal model. The level of total bilirubin, indirect bilirubin, brain bilirubin and brain iron was significantly increased in the modelling group. There was a significant decreased in all severity levels of the auditory brainstem response test in the two modelling group. Akinesia, bradykinesia and slip were significantly declined in the experience group. Apoptosis in basal ganglia and cerebellum were significantly decreased in the stem cell-treated group in comparison to the vehicle group. All severity levels of the auditory brainstem response tests were significantly decreased in 2-month-old rats. Transplantation results in the substantial alleviation of walking impairment, apoptosis and auditory dysfunction. This study provides important information for the development of therapeutic strategies using human adiposity-derived stem cells in prenatal brain damage to reduce potential sensori motor deficit.
Subject(s)
Adipose Tissue/cytology , Kernicterus/surgery , Stem Cell Transplantation/methods , Stem Cells/physiology , Animals , Animals, Newborn , Anti-Infective Agents/toxicity , Antigens, CD/metabolism , Brain/cytology , Brain/metabolism , Disease Models, Animal , Flow Cytometry , Humans , Iron/metabolism , Kernicterus/chemically induced , Kernicterus/complications , Male , Oxidants/toxicity , Phenylhydrazines/toxicity , Rats , Rats, Wistar , Sensory Gating/drug effects , Sulfisoxazole/toxicityABSTRACT
The chick micromass culture system has advantages over the validated rat system - ready availability and non-culling of the donor parent - but needs to give comparable results. This study confirmed comparability and the ability to extend the system to cover cardiac effects. It was also compared with the validated embryonic stem cell cardiomyocyte model. A teratogen and paired non-teratogen with known in vivo effects were used. Differential effects were measured via changes in cell protein content, cell viability (resazurin reduction and neutral red uptake), and cell contractility. Results showed that teratogens [L-ethionine, 5-fluorouracil and sulphisoxazole] could be distinguished from non-teratogens [DL-methionine, 6-methyluracil and sulphanilamide respectively]. Dichloroacetone and dichloropropanol affected embryonic stem cells but not the micromass; dichloropropanol had a greater effect than dichloroacetone. This approach revealed differential effects on contractility independent of effects on activity/viability, whilst the total cell protein remained unchanged. We suggest that pre-validation of this system should be examined.
Subject(s)
Animal Testing Alternatives , Cell Culture Techniques , Myocytes, Cardiac/cytology , Teratogens/toxicity , Toxicity Tests/methods , Acetone/analogs & derivatives , Acetone/toxicity , Animals , Cell Count , Cell Differentiation/drug effects , Cell Separation , Cell Survival/drug effects , Cells, Cultured , Chick Embryo , Culture Media/pharmacology , Dose-Response Relationship, Drug , Embryo, Nonmammalian , Embryonic Stem Cells/drug effects , Ethionine/toxicity , Fluorouracil/toxicity , Indicators and Reagents/metabolism , Methionine/toxicity , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Neutral Red/metabolism , Organic Chemicals/metabolism , Oxazines/analysis , Sulfanilamide , Sulfanilamides/toxicity , Sulfisoxazole/toxicity , Time Factors , Uracil/analogs & derivatives , Uracil/toxicity , Xanthenes/analysis , alpha-Chlorohydrin/analogs & derivatives , alpha-Chlorohydrin/toxicityABSTRACT
The Syrian hamster embryo (SHE) cell transformation assay evaluates the potential of chemicals to induce morphological transformation in karyotypically normal primary cells. Induction of transformation has been shown to correlate well with the carcinogenicity of many compounds in the rodent bioassay. Historically the assay has not received wide-spread use due to technical difficulty. An improved protocol for a low pH 6.7 assay was developed by LeBoeuf et al. [R.A. LeBoeuf, G.A. Kerckaert, M.J. Aardema, D.P. Gibson, R. Brauninger, R.J. Isfort, Mutat. Res., 356 (1996) 85-127], that greatly reduced many of the technical difficulties associated with the SHE assay. The purpose of this paper is to describe the most current execution of the pH 6.70 protocol including protocol refinements made since the publication of a comprehensive protocol for this assay in Kerckaert et al. [G.A. Kerckaert, R.J. Isfort, G.J. Carr, M.J. Aardema, Mutat. Res., 356 (1996) 65-84].
Subject(s)
Carcinogenicity Tests/methods , Animals , Cell Transformation, Neoplastic , Cricetinae , Cyclophosphamide/toxicity , Cyclosporine/toxicity , Embryo, Mammalian , Hydrogen-Ion Concentration , Mesocricetus , Sulfisoxazole/toxicityABSTRACT
The effect of therapeutic concentration of sulfisuxazole (sulfa) on the bilirubin uptake and viability of 8402 cells in culture was studied. The total bilirubin was kept constant at 24 microM and albumin was added to obtain bilirubin-albumin molar ratio (BAMR) ranging from 0.5 to 2. The effect of sulfa on unbound bilirubin (UB) was measured by the peroxidase assay. Sulfa increased UB at all BAMR's more so at 1.5 and 2.0 (P less than 0.05). Significant increase in bilirubin uptake/cell with a corresponding decrease in cell viability was noted with sulfa at BAMR's greater than 1 (P less than 0.05). The LD50 of these cells was 73-86 nM of UB. A plot of cell viability versus UB for the combined control and sulfa data (r2 = 0.94) confirms that the decrease in cell viability with increasing BAMR is explained by the corresponding rise in UB. Therefore, increase in UB by sulfa leads to toxicity of 8402 cells. Thus, we speculate that they may be used to study the mechanism of bilirubin neurotoxicity induced by similar drugs.
