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Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human
D Y Hwang1, K R Chae, D H Shin
1Division of Laboratory Animal Resources, Korea FDA, National Institute of Toxicological Research, Seoul, 122-704, Korea.
Abstract:
The cytochrome P450 enzymes (P450s or CYPs) are a superfamily of hemeproteins that catalyze the monooxygenation of a wide range of endobiotic and xenobiotic substrates. A typical strategy in toxicological research and testing involves applying a toxicant at high doses for a short period to homogeneous animals under controlled conditions. However, the conditions of this approach have very little in common with actual human exposure. Transgenic (Tg) mice carrying human genes encoding a drug-metabolizing enzyme (CYP) offer a solution to many of the difficulties in the evaluation of chemical toxicity. It has been demonstrated that the expression of human CYP transgenes under the control of mammalian-inducible promoters exhibits relatively poor fold increases after induction. In this study, we used the tetracycline-regulated (tet) promoter system to increase the expression of the human CYP1B1 (hCYP1B1) gene in the tissues of transgenic mice. By mating two lineages of transgenic mice, double transgenic (dTg) mice expressing both tTA and hCYP1B1 genes under the control of the tet promoter were successfully produced, into which the two transgenes were introduced in an embryo. The expression pattern of tTA-driven hCYP1B1 transgene featured a fold induction of more than 3 to 12 in the brain, heart, and lung and 2- to 4-fold induction in the liver, kidney, and intestine upon doxycycline removal. Immunohistochemical staining with hCYP1B1 antibody was also increased by the removal of doxycycline. In addition, the activities of CYP liver microsomes in the dTg mice without doxycycline showed an increase compared to that in the dTg mice treated with doxycycline. The level of activities correspond to the levels of human CYP1B1 protein expression in the Tg mice (-dox) that was increased by 2-fold induction as compared to that of the dTg mice with doxycycline. Thus, overproduction in Tg can be purified and the activity of purified human CYP1B1 can be characterized by alterations to the coding sequence in order to solve the physiological function of this enzyme in a humanized in vivo system. It is also possible to examine the activity of purified human CYP1B1 using several environmental toxicants such as procarcinogens.
Insights
Researchers developed a novel transgenic mouse model to better study human drug metabolism and toxicity. This model uses a tetracycline-regulated system to control the expression of human CYP1B1, enabling more accurate in vivo toxicological assessments.
Area of Science:
- Pharmacology and Toxicology
- Genetics and Molecular Biology
- Biochemistry
Background:
- Cytochrome P450 enzymes (CYPs) are crucial for metabolizing diverse compounds.
- Traditional toxicological studies using high-dose, short-term animal exposures poorly mimic human environmental exposures.
- Transgenic (Tg) mice expressing human CYPs offer a more relevant model, but achieving significant transgene induction has been challenging.
Purpose of the Study:
- To enhance the expression of the human CYP1B1 (hCYP1B1) gene in transgenic mice using a tetracycline-regulated (tet) promoter system.
- To create a double transgenic (dTg) mouse model for improved in vivo evaluation of chemical toxicity and drug metabolism.
- To investigate the physiological function and activity of human CYP1B1 in a humanized in vivo system.
Main Methods:
- Developed double transgenic (dTg) mice by mating two lineages, one expressing tTA and the other expressing hCYP1B1 under the tet promoter.
- Administered doxycycline to regulate transgene expression and subsequently removed it to induce hCYP1B1 expression.
- Quantified hCYP1B1 expression via immunohistochemistry and measured CYP liver microsomal activity in dTg mice with and without doxycycline.
Main Results:
- Doxycycline removal led to significant fold induction of hCYP1B1 transgene expression (3-12 fold in brain, heart, lung; 2-4 fold in liver, kidney, intestine).
- Immunohistochemical staining confirmed increased hCYP1B1 protein levels upon doxycycline removal.
- CYP liver microsomal activity increased in dTg mice without doxycycline, correlating with elevated hCYP1B1 protein levels.
Conclusions:
- The tetracycline-regulated system effectively enhances hCYP1B1 expression in transgenic mice, creating a valuable humanized in vivo model.
- This dTg mouse model facilitates the purification and characterization of human CYP1B1 activity, aiding in understanding its physiological role.
- The model enables the assessment of environmental toxicants, such as procarcinogens, using purified human CYP1B1 activity.
