Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.     

CYP3A4 and pregnane X receptor humanized mice

Marked species differences exist in P450 expression and activities. In order to produce mouse models that can be used to more accurately predict human drug and carcinogen metabolism, P450- and xenobiotic receptor humanized mice are being prepared using bacterial artificial chromosomes (BAC) and P1 phage artificial chromosomes (PAC) genomic clones. In some cases, transgenic mice carrying the human genes are bred with null-mice to produce fully humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 were generated and characterized. Studies with the CYP3A4-humanized (hCYP3A4) mouse line revealed new information on the physiological function of this P450 and its role in drug metabolism in vivo. With this mouse line, CYP3A4, under certain circumstances, was found to alter the serum levels of estrogen resulting in deficient lactation and low pup survival as a result of underdeveloped mammary glands. This hCYP3A4 mouse established the importance of intestinal CYP3A4 in the pharmacokinetics of orally administered drugs. The hCYP3A4 mice were also used to establish the mechanisms of potential gender differences in CYP3A4 expression (adult female > adult male) that could account for human gender differences in drug metabolism and response. The pregnane X receptor (PXR) is also involved in induction of drug metabolism through its target genes including CYP3A4. Since species differences exist in ligand specificity between human and mice, a PXR-humanized mouse (hPXR) was produced that responds to human PXR activators such as rifampicin but does not respond to the rodent activator pregnenalone 16alpha-carbonitrile.     

The human CYP1A1 gene is regulated in a developmental and tissue-specific fashion in transgenic mice

Regulation and expression of human CYP1A1 is demonstrated in transgenic mice. We have developed two transgenic mouse lines. One mouse strain (CYPLucR) carries a functional human CYP1A1 promoter (-1612 to +293)-luciferase reporter gene, and the other strain (CYP1A1N) expresses CYP1A1 under control of the full-length human CYP1A1 gene and 9 kb of flanking regulatory DNA. With CYPLucR(+/-) mice, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and several other aryl hydrocarbon receptor ligands induced hepatocyte-specific luciferase activity. When other tissues were examined, TCDD induced luciferase activity in brain with limited induction in lung and no detectable luciferase activity in kidney. Treatment of CYP1A1N(+/-) mice with TCDD resulted in induction of human CYP1A1 in liver and lung, while mouse Cyp1a1 was induced in liver, lung, and kidney. Although induced CYP1A1/Cyp1a1 could not be detected by Western blot analysis in brains from CYP1A1N(+/-) mice, induction in brain was verified by detection of CYP1A1/Cyp1a1 RNA. The administration of TCDD to nursing mothers to examine the effect of lactational exposure via milk demonstrated prominent induction of luciferase activity in livers of CYPLucR(+/-) newborn pups with limited induction in brain. However, TCDD treatment of adult CYPLucR(+/-) mice led to a 7-10-fold induction of brain luciferase activity. Combined these results indicate that tissue-specific and developmental factors are controlling aryl hydrocarbon receptor-mediated induction of human CYP1A1.     

Repair of thymine glycol by hNth1 and hNeil1 is modulated by base pairing and cis-trans epimerization

Oxidation of thymine yields 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol. Tg) which, as cis 5S,6R and 5R,6S 2'-deoxyribonucleoside diastereoisomers (dTg1, dTg2), are in equilibrium with their trans 5S,6S and 5R,6R epimers. The stereoselective excision of Tg from DNA by the mammalian orthologs of E. coli DNA N-glycosylase/AP lyases Nth and Nei was reported using substrates in which Tg opposed adenine. Since we showed that Tg is the major product of oxidation of 5-methylcytosine, we asked if the opposing purine influenced stereospecific enzymatic excision. The human ortholog hNth1 released Tg2 much more rapidly than Tg1 regardless of the opposing purine. In contrast, hNeil1 released Tg non-stereoselectively, but the rate of excision was much greater when Tg opposed guanine. Remarkably, the kinetics of excision of Tg by hNth1 and hNeil1 were biphasic, describing a double exponential curve which yielded two rate constants. We suggest that the greater rate constant describes the rate of enzymatic excision of Tg. The smaller rate constant represents the equilibrium constant for the cis and trans epimerization of dTg1 and dTg2 in high molecular weight DNA. Thus, only one of the epimers of dTg1 and dTg2 are enzymatically processed but it is not yet known whether it is cis or trans. Thus, base excision repair of Tg in mammals is mediated by at least two DNA N-glycosylase/AP lyases which are affected by the nature of the diastereoisomer of dTg, the rate of cis-trans epimerization of each diastereoisomer, and the nature of the opposing purine.     

B Cell Activating Factor (BAFF) and T Cells Cooperate to Breach B Cell Tolerance in Lupus-Prone New Zealand Black (NZB) Mice

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.     

A transgenic mouse expressing human CYP4B1 in the liver

The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene. Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay. These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody. However, such inhibition was not observed in control mice. This is the first report to indicate catalytic activities of human CYP4B1. For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells. It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid. This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis.     

Elevated hepatic and depressed renal cytochrome P450 activity in the Tg2576 transgenic mouse model of Alzheimer's disease

Recent studies indicate that the Tg2576 transgenic mouse model of Alzheimer's disease [tg(hAPP)] demonstrates disturbances in plasma glucose and neuroendocrine function reminiscent of Alzheimer's disease (AD). Alterations in any one of these systems can have a profound effect on hepatic cytochrome P450 (CYP) expression. Additionally, the recent discovery that amyloid beta 1-42 can induce the expression of CYP reductase in neuronal cultures further suggests that hepatic CYP-related metabolism may be affected by the expression of mutant human amyloid precursor protein in these tg(hAPP) mice. Therefore, the current study was conducted to investigate the activity and protein content of several CYP isoforms in the livers and kidneys of aged (20-month-old) tg(hAPP) mice. tg(hAPP) mice exhibit significant elevations in hepatic CYP2B, CYP2E1-, CYP3A- and CYP4A-associated activities and CYP4A immunoreactive protein compared with wild-type. In contrast to the liver, a significant depression in renal CYP2E1- and CYP4A-associated activities were demonstrated in tg(hAPP) mice. The presence of the mutant hAPP protein was detected in the brain, kidney and livers of tg(hAPP) mice.     

Conditional expression of fibroblast growth factor-7 in the developing and mature lung

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)(7)-cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta x (Teto)(7)-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.     

Adipose Overexpression of Heme Oxygenase-1 Does Not Protect against High Fat Diet-Induced Insulin Resistance in Mice

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme with potent anti-oxidant and anti-inflammatory activities. Previous studies have shown that systemic induction of HO-1 by chemical inducers reduces adiposity and improves insulin sensitivity. To dissect the specific function of HO-1 in adipose tissue, we generated transgenic mice with adipose HO-1 overexpression using the adipocyte-specific aP2 promoter. The transgenic (Tg) mice exhibit similar metabolic phenotype as wild type (WT) control under chow-fed condition. High fat diet (HFD) challenge significantly increased the body weights of WT and Tg mice to a similar extent. Likewise, HFD-induced glucose intolerance and insulin resistance were not much different between WT and Tg mice. Analysis of the adipose tissue gene expression revealed that the mRNA levels of adiponectin and interleukin-10 were significantly higher in chow diet-fed Tg mice as compared to WT counterparts, whereas HFD induced downregulation of adiponectin gene expression in both Tg and WT mice to a similar level. HFD-induced proinflammatory cytokine expression in adipose tissues were comparable between WT and transgenic mice. Nevertheless, immunohistochemistry and gene expression analysis showed that the number of infiltrating macrophages with preferential expression of M2 markers was significantly higher in the adipose tissue of obese Tg mice than WT mice. Further experiment demonstrated that myeloid cells from Tg mice expressed higher level of HO-1 and exhibited greater migration response toward chemoattractant in vitro. Collectively, these data indicate that HO-1 overexpression in adipocytes does not protect against HFD-induced obesity and the development of insulin resistance in mice.     

O2 enhancement of human trophoblast differentiation and hCYP19 (aromatase) gene expression are mediated by proteasomal degradation of USF1 and USF2

When cultured in 20% O(2), human cytotrophoblasts fuse to form the syncytiotrophoblast with marked induction of hCYP19 (aromatase) gene expression. When cultured in 2% O(2), cytotrophoblast fusion and induced hCYP19 expression are prevented. These effects of hypoxia are mediated by increased expression of mammalian achaete/scute homologue-2 (Mash-2), which increases levels of upstream stimulatory factors 1 and 2 (USF1/2) and their binding as heterodimers to E-boxes surrounding the hCYP19 promoter. In studies to define mechanisms for O(2) regulation of syncytiotrophoblast differentiation, we found that hypoxia and overexpression of Mash-2 markedly increased cyclin B1 levels in cultured trophoblasts and the proportion of cells at the G(2)/M transition. Unlike USF proteins, USF1/2 mRNA levels are unaffected by O(2) tension. To determine whether increased O(2) might enhance proteasomal degradation of USF1/2, human trophoblasts were cultured in 2% or 20% O(2) with or without proteasome inhibitors. In cells cultured in 20% O(2), proteasome inhibitors increased USF1/2 protein levels and blocked spontaneous induction of hCYP19 expression, cell fusion, and differentiation. Like hypoxia, inhibitory effects of proteasome inhibitors on hCYP19 expression were mediated by increased binding of USF1/2 to the E-boxes. In human trophoblast cells cultured in 20% O(2), increased polyubiquitylation of USF1/2 proteins was observed. Thus, early in gestation when the placenta is relatively hypoxic, increased USF1/2 may block trophoblast differentiation and hCYP19 gene expression. In the second trimester, increased O(2) tension promotes proteasomal degradation of USF1/2, resulting in syncytiotrophoblast differentiation and induction of hCYP19 expression.     

Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1

Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glu-cobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4-methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4-methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.     

The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer

In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.     

Constitutive expression and induction of CYP1B1 mRNA in the mouse.

Previous studies appeared to indicate that CYP1B1 was not constitutively expressed in mouse liver. In our laboratory, we demonstrated using aromatic hydrocarbon-responsive receptor knock-out (AHR-(-)/-) mice that both piperonyl butoxide (PBO) and acenaphtyhlene (ACN) are AHR-independent inducers of murine CYP1A2 and CYP1B1 mRNA. In the current study, we demonstrate both constitutive levels and induction of CYP1B1 in mouse liver. The induction of CYP1B1 mRNA by PBO or ACN was higher in DBA/2 (Ahrd) than in C57BL/6 (Ahrb-1) mice, while 3-methylcholanthrene induced CYP1B1 more in C57BL/6 than in DBA/2 mice. These results suggest that CYP1B1 may also be induced by more than one mechanism. In addition, constitutive expression of CYP1B1 was detected in liver, kidney, and lung of untreated C57BL/6 mice. There was no gender difference in CYP1B1 expression; however, in C57BL/6 mice, the kidney contained less CYP1B1 than either liver or lung.