1. Application Of Cardiac Specific CYP2E1 Gene Modified Mice In Preclinical Cardiac Toxicity Evaluation 2. Establishment Of Guinea Pig Nickel Allergic Contact Dermatitis Model

Many drugs developed by the pharmaceutical industry fail in drugs research and development stage because of unanticipated toxic side effects. Toxicity may be caused by a compound interacting with a molecular target that is distinct from target selected for therapeutic benefits, defined as an off-target effect. Toxicity continues to be a major factor in the attrition of candidate drugs. A recent analysis of nonclinical drug candidate attrition suggests areas of particular concern include cardiovascular toxicity (26%). With the development of science and technology, computer-aid drug design and other new technology, the number of new chemical substances increase sharply. An important strategy for lowering drug development costs involves termination of compounds as early in the process as possible, before substantial resources have been invested in clinical trials.Drugs are usually tested for in vivo toxicity in mammalian models, such as mouse, rat and dog. And vitro assays are amenable to high-throughput screening, but have been hampered by the unexpected difficulty of correlating observed transcriptional profiles with known mechanisms of toxicity as well as a lack of consensus around approaches and study designs. Cell-based assays are also limited in their ability to mirror the metabolism of a whole animal and to accurately model multi-cellular processes. However, traditional toxicity testing is a time-consuming and expensive part of the modern drug discovery process, making them poorly suited for large-scale toxicology screening. Furthermore, the diseases due to the drug adverse reaction have evolved into a serious clinical problem. So, it’s very significant to identify exactly the relation between the patient reaction and the drug during new drug development or post-marketing evaluation. Transgenic animal is a hotspot of this research. The target gene that genetically engineered into animal’s DNA genome is expected to own convenience to evaluate drugs.Cytochrome P4502E1(CYP2E1) is a cytochrome P450enzyme that catalyzes the metabolism of toxic substrates. The expression of this enzyme is marked in the liver, heart, lungs, pancreas, brain and intestine. It is expressed immediately after birth and is maximally transcribed within the first week. The expression of CYP2E1is increased in the human ischemic and dilated heart and in left ventricular tissue of the spontaneously hypertensive rats. CYP2E1-catalyzed metabolism may also cause toxicity or cell damage through the production of toxic metabolites, oxygen radicals and lipid peroxidation.In our study, two kinds of genetically modified mouse (a heart tissue-specific overexpression CYP2E1transgenic mice-α-MHC CYP2E1transgenic mice (Tg) and a heart tissue-specific CYP2E1silence mice-α-MHC CYP2E1silence mice (sTg)) were used to assess the specificity and sensibility of those two kinds of animal in the cardiotoxicity drugs evaluation. At first, enough mice were bred and genotyped the PCR using respective primers. The weight and food intake of those two kinds of transgenic mice at different age is systematically detected and collected to provide the fundamental date for the deep research of the evaluation of new drug safety. Then, with the use of Tg and sTg mice as models, we performed experiments to study the cardiotoxicity of acetaminophen (APAP), a specific metabolic substrate of CYP2E1. We found that the modifying of CYP2E1gene in heart tissue can not affect the affinity of APAP in heart. Compared with the wild type of C57BL/6mice, the cardiotoxicity of APAP is not obvious in Tg and sTg mice. Doxorubicin (DOX), an anthracycline antibiotic, is a potent spectrum chemotherapeutic agent that is effective against a variety of human malignancies. However, the clinical use of DOX is limited by its severe, cumulative, dose-dependent cardiotoxicity. And various mechanisms of DOX cardiotoxicity has been proposed, the most commonly acknowledged view is the free radical theory of injury. Last but not least, the cardiotoxicity of DOX was evaluated from the aspects of systematical biology. The results from biomedical examination prove the appearance of cardiotoxic reaction in all DOX treated mice. We found that the cardiotoxicity was more severe in Tg mice groups. From those results, α-MHC CYP2E1transgenic mice might be a good try for cardiac toxicity evaluation. But more evidence should be collected. Objective To establish a guinea-pig model of nickel allergic contact dermatitis (ACD) and study the significance of several factors (such as the concentration of nickel, duration of exposure, cutaneous microenvironment and the schedule of elicitation) for the nickel ACD model. Methods Eighty-six guinea-pigs (230-270g) were divided randomly into fourteen groups by the different factors (the concentration of nickel, duration of exposure, cutaneous microenvironment and the schedule of elicitation); The groups of20%Ni-28d-6h、10%Ni-28d-6h、20%Ni-28d-SP-6h、10%Ni-28d-SP-6h、20%Ni-28d-24h、10%Ni-28d-24h、20%Ni-28d-SP-24h and10%Ni-28d-SP-24h were sensitized in day0,7,14and challenged in day28with nickel sulfate and the groups of20%Ni-9d-6h、20%Ni-9d-24h、20%Ni-9d-SP-6h、20%Ni-9d-SP-24h、 positive control (PC) and negative control (NC) were sensitized in day0,1,2and challenged in day9. Dynamic grading of the skin alterations on the skin was performed at6,12,24,48and72h after challenged. ELISA was used to measure the levels of IL-2and IFN-y in the guinea-pig’s skin of group20%Ni-9d-6h、20%Ni-9d-24h、20%Ni-9d-SP-6h、20%Ni-9d-SP-24h、PC and NC. H.E staining was used for the pathological examination. Results The most severe erythema was observed at48h in the groups of day28， but at24h in the groups of day9. And the swelling was not obvious in all the guinea pigs. The levels of IL-2and IFN-y in the skin were up to the peak (IL-2533.53±41.51) pg/ml, IFN-y (1235.63±76.85) pg/ml) in the groups of day9and until72h. There were amount of inflammatory cells infiltrating, escharosis, hyperkeratosis, angiotelectasis in the epidermis and dermis in all the animals. Those showed they behaved in the similar way as human ACD. Conclusions The guinea-pig models of nickel ACD have been successfully established for different situations in the clinical research. The more nickel and duration of exposure is, the worse skin reaction gets. Synthetic perspiration (SP) in the nickel and the schedule of sensitizing in day0,1,2and challenging in day9can also get the same ending.