Imipramine Induces Apoptosis and Inhibits the Metastatic Potential of Triple-negative Breast Cancer Cells.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is an aggressive and deadly subtype of breast cancer, and there is an urgent need for new therapeutic strategies. The highly metastatic and anti-apoptotic characteristics are known to be the major factors causing uncontrolled growth in TNBC. Imipramine is a tricyclic antidepressant that possesses anti-inflammatory activity and has been reported to inhibit the progression of highly metastatic non-small cell lung cancer. MATERIALS AND METHODS: This study used MTT assay, apoptosis markers flow cytometry analysis, open-source data analysis, NF-B reporter gene assay, and western blotting to elucidate the effect of imipramine on MDA-MB-231 and 4T1 cells. RESULTS: Imipramine induced caspase-mediated extrinsic and intrinsic apoptosis and was potentially associated with patient overall survival. Furthermore, imipramine suppressed the invasion and migration abilities and the expression of metastasis-associated proteins in TNBC cells. CONCLUSION: Imipramine effectively suppressed TNBC progression by inducing apoptosis and inhibiting metastasis.

Accessing Apoptosis Induction and Metastasis Inhibition Effect of Magnolol on Triple Negative Breast Cancer In Vitro.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer that still requires improvement in treatment. Magnolol extract, derived from the bark of Magnolia officinalis, has traditionally been used in Asia to treat sleeping disorders and anxiety, and as an anti-inflammatory agent. Several reports have indicated that magnolol may have the potential to inhibit the progression of hepatocellular carcinoma and glioblastoma. However, the anti-tumor effect of magnolol on TNBC remains unknown. MATERIALS AND METHODS: In this study, we used two TNBC cell lines, MDA-MB-231 and 4T1, to examine the cytotoxicity, apoptosis, and metastasis effects of magnolol. These were evaluated using MTT assay, flow cytometry, western blotting, and invasion/migration transwell assay, respectively. RESULTS: Magnolol significantly induced cytotoxicity and extrinsic/intrinsic apoptosis in both TNBC cell lines. It also decreased metastasis and associated protein expression in a dose-dependent manner. Furthermore, the anti-tumor effect was associated with the inactivation of the epidermal growth factor receptor (EGFR)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT3) signaling pathway. CONCLUSION: Magnolol may not only induce cell death in TNBC through apoptosis signaling activation but also by down-regulating EGFR/JAK/STAT3 signaling, which mediates TNBC progression.

Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts.

Most cases of early onset torsion dystonia (DYT1) are caused by a 3-base pair deletion in one allele of the TOR1A gene causing loss of a glutamate in torsinA, a luminal protein in the nuclear envelope. This dominantly inherited neurologic disease has reduced penetrance and no other medical manifestations. It has been challenging to understand the neuronal abnormalities as cells and mouse models which are heterozygous (Het) for the mutant allele are quite similar to wild-type (WT) controls. Here we found that patient fibroblasts and mouse neurons Het for this mutation showed significant differences from WT cells in several parameters revealed by infection with herpes simplex virus type 1 (HSV) which replicates in the nucleus and egresses out through the nuclear envelope. Using a red fluorescent protein capsid to monitor HSV infection, patient fibroblasts showed decreased viral plaque formation as compared to controls. Mouse Het neurons had a decrease in cytoplasmic, but not nuclear HSV fluorescence, and reduced numbers of capsids entering axons as compared to infected WT neurons. These findings point to altered dynamics of the nuclear envelope in cells with the patient genotype, which can provide assays to screen for therapeutic agents that can normalize these cells.

The prescribing trend of oral antidiabetic agents for type 2 diabetes in Taiwan: An 8-year population-based study.

The purpose of this study was to evaluate the prescription trend and pattern of oral antidiabetic (OAD) medications, which are extensively used worldwide for treating type 2 diabetes, in 2 age groups.In this population-based study, data obtained from the National Health Insurance Research Database, Taiwan, were analyzed to investigate the prescription trend of all types of OAD medications during 2005 to 2012. We used descriptive statistics to demonstrate the trend of prescription patterns stratified by age (aged 65 years and above or younger than 65).Sulfonylurea (SU) was once the most commonly used drug, but the proportion of its prescription had declined gradually (76.83% in 2005 to 63.70% in 2012). Consequently, biguanide (BG) became the most commonly used drug since 2010 (64.31% in 2005 to 74.41% in 2012). In addition, the prescriptions of thiazolidinedione decreased significantly (9.20% in 2005 to 2.86% in 2012), whereas the usage of DPP-4 inhibitor increased with time (3.73% in 2009 to 19.64% in 2012). The treatment choice of SU and α-glucosidase inhibitor (AGI) was higher in elderly patients compared with the younger population (SU: 62.70% in 2012, AGI: 12.78% in 2012). Two-drug combination therapies were the prevalent treatment choices for patients with type 2 diabetes (44.77% in 2012), particularly in the elderly group; however, ≥3 drug combination therapies increased gradually during the study period, particularly in the younger group.This descriptive study presents the change in the prescription of OAD medication for different age groups during 2005 to 2012.

Correlation of the experimental and numerical results for the holding power of dental, traumatic, and spinal screws.

The holding power of the bone-screw interfaces is one of the key factors in the clinical performance of screw design. The value of the holding power can be experimentally measured by pullout tests. Historically, some researchers have used the finite-element method to simulate the holding power of the different screws. Among them, however, the assumed displacement of the screw withdrawal is unreasonably small (about 0.005-1.0 mm). In addition, the chosen numerical indices are quite different, including maximum stress, strain energy, and reaction force. This study systematically uses dental, traumatic, and spinal screws to experimentally measure and numerically simulate their bone-purchasing ability within the synthetic bone. The testing results (pullout displacement and holding power) and numerical indices (maximum stress, total strain energy, and reaction forces) are chosen to calculate their correlation coefficients. The pullout displacement is divided into five regions from initial to final withdrawal. The experimental results demonstrate that the pullout displacement consistently occurs at the final region (0.6-1.6 mm) and is significantly higher than the assumed value of the literature studies. For all screw groups, the measured holding power within the initial region is not highly or even negatively correlated with the experimental and numerical results within the final region. The observation from the simulative results shows the maximum stress only reflects the loads concentrated at some local site(s) and is the least correlated to the measured holding power. Comparatively, both energy and force are more global indices to correlate with the gross failure at the bone-screw interfaces. However, the energy index is not suitable for the screw groups with rather tiny threads compared with the other specifications. In conclusion, the underestimated displacement leads to erroneous results in the screw-pullout simulation. Among three numerical indices the reaction-force is the optimal index for the screw-pullout problem.

Curcumin blocks migration and invasion of mouse-rat hybrid retina ganglion cells (N18) through the inhibition of MMP-2, -9, FAK, Rho A and Rock-1 gene expression.

Cancer metastasis involves multiple processes which may complicate clinical management and even lead to death. Matrix metalloproteinases (MMPs) play an important role in cancer cell invasion, metastasis and angiogenesis, depending on whether agents can inhibit MMPs which could lead to inhibition of the migration and invasion of cancer cells. Curcumin, the active constituent of the dietary spice turmeric, has potential for the prevention and therapy of cancer. However, there is no study to address the effects of curcumin on migration and invasion of mouse-rat hybrid retina ganglion cells (N18). This is the first study to explore the anti-migration and -invasion of curcumin in mouse-rat hybrid retina ganglion cells (N18) in vitro. Curcumin exerted a dose- and time-dependent inhibitory effect on the invasion and migration of N18 cells in vitro. Results from Western blotting showed that curcumin inhibited the protein levels of PKC, FAK, NF-kappaB p65 and Rho A leading to the inhibition of ERK1/2, MKK7, COX-2 and ROCK1, respectively, finally causing the inhibition of MMP-2 and -9 for the inhibition of migration and invasion of N18 cells. Moreover, this action was involved in the inhibition of gene expression of MMP-2 and -7, FAK, ROCK1 and Rho A. Overall, the above data show that the anticancer effect of curcumin also exists for the inhibition of migration and invasion in N18 cells, and that curcumin may be a powerful candidate for developing preventive agents for cancer metastasis.

Primary cutaneous diffuse large B cell lymphoma relapsed solely as a huge lung tumor mimicking a primary pulmonary lymphoma.

Primary cutaneous large B cell lymphoma, leg type (PCLBCL-leg) has recently been identified and recognized as a specific entity. Patients with PCLBCL-leg have a higher relapse rate and a poorer prognosis than the other types of primary cutaneous B cell lymphomas, and disease relapse is confined to the skin in the majority of cases with rare exclusive extracutaneous progression. The late occurrence of lymphoma in patients with a prior history of lymphoma may represent a relapse/progression or a distinct tumor unrelated to the original one. The distinction is of important clinical and therapeutic implications. Here, we report the case of a 90-year-old lady with a history of PCLBCL-leg in complete remission after radiotherapy that developed a huge, solitary pulmonary lymphoma without lymphadenopathy 14 months later. The latter was initially considered as stage IE primary pulmonary lymphoma and was treated with combination chemotherapy resulting in complete remission. Retrospective pathologic review and B cell clonality study revealed that the pulmonary tumor was a diffuse large B cell lymphoma of the same clonal origin as the PCLBCL-leg. This case is unique in the exclusive pulmonary relapse and illustrates the importance of expert pathological review and molecular study in the management of lymphoma patients with unusual clinical features.

Extramammary Paget's disease of the scrotum associated with hepatocellular carcinoma.

Extramammary Paget's disease (EMPD) is a rare cutaneous carcinoma of epidermal origin. The diagnosis is frequently delayed, and the disease tends to be associated with an underlying adnexal or internal malignancy. There have been several reports of EMPD associated with carcinoma of the bladder, prostate, kidney, and colon. The association of hepatocellular carcinoma (HCC) with EMPD appears to be exceedingly rare; to our knowledge, it has been reported only once in the English literature. Herein, we report an unusual case of EMPD of the scrotum associated with HCC. EMPD was diagnosed 1 year after the appearance of an erythematous plaque, and HCC was noted 19 months after the diagnosis of EMPD. From our experience and literature review, in patients with nonspecific skin lesions that are unresponsive to conventional treatment, EMPD should be considered and skin biopsy performed. Long-term follow-up is needed to watch for the appearance of adnexal carcinoma or internal malignancy.

Baicalein-induced apoptosis via endoplasmic reticulum stress through elevations of reactive oxygen species and mitochondria dependent pathway in mouse-rat hybrid retina ganglion cells (N18).

Studies were designed to investigate the effects of baicalein on mouse-rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, reactive oxygen species (ROS), cytoplasmic Ca2+, mitochondrial membrane potential (MMP), apoptosis induction, and caspases-3 activity were examined by flow cytometric assay. Apoptosis-associated proteins such as p53, Bax, Bcl-2, cytochrome c, and caspase-3 were examined by Western blot. We demonstrated the increase in the levels of p53, Bax, and cytochrome c and decrease in the level of Bcl-2, which are associated with the induction of apoptotic cell death after 24 h treatment with baicalein in N18 cells. Baicalein induced an increase in the cytoplasmic levels of ROS and Ca2+ in 1 h and reached their peak at 3 h, and thereafter a loss of MMP by flow cytometry. We also demonstrated a release of the cytochrome c from mitochondria into cytosol and an activation of caspase-3, which led to the occurrence of apoptosis in N18 cells treated with baicalein by Western blot. Pretreatment was conducted with BAPTA (intracellular calcium chelator) in baicalein-treated cells, the decline of MMP was recovered, and the increase in the level of cytoplasmic Ca2+ was suppressed, and the proportion of apoptosis was also markedly diminished. In conclusion, our data suggests that oxidative stress and cellular Ca2+ modulates the baicalein-induced cell death via a Ca2+-dependent mitochondrial death pathway in N18 cells.

The role of Ca2+ in baicalein-induced apoptosis in human breast MDA-MB-231 cancer cells through mitochondria- and caspase-3-dependent pathway.

Baicalein was investigated for tumor cell-specific cytotoxicity, apoptosis-inducing activity and signal pathway against the MDA-MB-231 human breast cancer cell line. After the MDA-MB-231 cells had been treated with baicalein, trypan blue exclusion, propidium iodide (PI) assay and 4',6-diamidino-2-phenylindole (DAPI) were used to stain the dead cells and detect apoptosis, respectively. The effects of baicalein on the levels of reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (deltapsim) on MDA-MB-231 cells were examined by flow cytometric assays. The ROS caused endoplasmic reticulum (ER) stress, confirmed by the increase of GADD153 and GRP78 in the examined cells. GADD153 and GRP78 increases were also confirmed by confocal laser microscopy examination and indicated that both proteins translocated to the nucleus. The effects of baicalein on the expression of apoptotic-regulated genes, such as Bcl-2 family and caspase, were detected by Western blotting. To further investigate the apoptotic pathway and the role of Ca2+ induced by baicalein, a caspase-3 inhibitor and Ca2+ chelator were used to block caspase-3 activity and Ca2+ in MDA-MB-231 cells. Baicalein induced apoptosis in a time-dependent effect through the inhibition of Bcl-2 expression, increased the levels of Bax, reduced the level of deltapsim, and promoted the cytochrome c release and caspase-3 activation. MDA-MB-231 cells were pretreated with BAPTA which reduced the levels of Ca2+, deltapsim and apoptosis. In conclusion, baicalein induced apoptosis via Ca2+ production, mitochondria-dependent and caspase-3 activation in MDA-MB-231 cells.

Curcumin inhibits WEHI-3 leukemia cells in BALB/c mice in vivo.

Curcumin (1, 7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5- dione), a natural polyphenol product of the plant Curcuma longa, exhibited potent inhibitory activities against proliferation, induced cell cycle arrest and exhibited the induction of apoptosis in several tumor cell lines. In our previous studies, we have shown that curcumin induced cell cycle arrest and apoptosis on human leukemia HL-60 and mouse leukemia WEHI-3 cells; there are no reports regarding whether or not it affects leukemia cells in vivo. In the present study, we investigated the effects of curcumin on WEHI-3 in BALB/c mice and the results indicated that curcumin reduces the percentage of Mac-3 marker, which is the precursor of macrophage. Curcumin induced significant effects on the population of B cells from murine leukemia in vivo. We also investigated the weights of spleen and liver from murine leukemia and the results showed that curcumin reduced the weight of the liver and spleen. From the pathological examinations, the effects of curcumin on the liver and spleen from mice after being injected with WEHI-3 cells were apparent. Both organs were enlarged. In conclusion, curcumin affect WEHI-3 cells in vivo.

Diallyl disulfide induced signal transducer and activator of transcription 1 expression in human colon cancer colo 205 cells using differential display RT-PCR.

Diallyl disulfide is one of the components of garlic and has been demonstrated to induce apoptosis in many cancer cell lines, though it is not reported to be associated with signal transducer and activator of transcription 1 (STAT1) expression. Moreover the role of STAT1 does not directly affect apoptosis in cancer cells after exposure to chemotherapy agents, though some reports showed that STAT1 is associated with apoptosis. In this study, differential display RT-PCR was used to examine the effects of diallyl disulfide (DADS) on human colon cancer cells (colo 205). The results demonstrated that DADS induced the expression of STAT1 which was also confirmed using Western blotting. STAT1 decoy oligonucleotides were also used to block STAT1 mRNA and led to a decrease in the levels of STAT1 and to subsequence decrease in the percentage of apoptosis induced by DADS in examined colo 205 cells.

The role of Ca2+ on the DADS-induced apoptosis in mouse-rat hybrid retina ganglion cells (N18).

Diallyl disulfide (DADS), a component of garlic, has been shown to induce growth inhibition and apoptosis in human cancer cell types. The present studies were designed to investigate the effects of DADS on mouse-rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, cell cycle analysis, reactive oxygen species (ROS), Ca2+ production, mitochondria membrane potential, apoptosis induction, associated gene expression and caspases-3 activity were examined by flow cytometric assay and/or Western blot. After 24-h treatment with DADS, a dose- and time-dependent decrease in the viability of N18 cells was observed and the approximate IC50 was 27.6 microM. The decreased percentage of viable cells are associated with the production of ROS then followed by the production of Ca2+ which is induced by DADS. DADS induced apoptosis in N18 cells via the activation of caspase-3. DADS increased the protein levels of p53, cytochrome c and phosphated JNK within 24 h of treatment and it decreased the levels of Bcl-2 and those factors may have led to the mitochondria depolarization of N18 cells. DADS induced apoptosis were accompanied by increased levels of Ca2+ and decreased mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-3. Deleted levels of Ca2+ by BAPTA-AM 10 microM (intracellular calcium chelator) then led to decrease DADS-induced apoptosis. Inhibition of caspase-3 activation by inhibitor (z-VAD-fmk) completely blocked DADS-induced apoptosis on N18 cells. The results indicated that oxidative stress modulates cell proliferation and Ca2+ modulates the cell death induced by DADS.

Ketoprofen-inhibited N-acetyltransferase activity and gene expression in human colon tumor cells.

The activation of ketoprofen, which inhibits the outgrowth of azoxymethane-induced aberrant crypt foci in the rat colon, on the inhibition of arylamine N-acetyltransferase (NAT) activity (N-acetylation of substrates), gene expression (mRNA NAT) and 2-aminofluorene (AF)-DNA adduct formation was studied in a human colon tumor (adenocarcinoma) cell line (colo 205). Cellular cytosols (9000 xg supernatant) and intact colon tumor cells were used. The NAT activity in colo 205 cells was inhibited by ketoprofen in a dose- and time -dependent manner in both examined systems. The data also indicated that ketoprofen decreased the apparent value of V(max) of NAT enzymes, being a competitive inhibitor of NAT enzymes. The AF-DNA adduct formation in colo 205 cells was also decreased by ketoprofen. Based on the results from PCR, it was shown that ketoprofen affected mRNA NAT expression in human colon colo 205 cells. The cells were stained with anti-NAT antibody, then analyzed by flow cytometry. The results showed that ketoprofen decreased the percentage of cells stained by anti-NAT. This report is the first to demonstrate that ketoprofen inhibits human colon tumor cell NAT activity, gene expression and DNA adduct formation.

Cloning and immunolocalization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes.

A 30-kDa protein extracted from the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes has been identified as a thermostable chitinase based on its enzymatic activity. A cDNA fragment encoding the precursor protein (including a cleavable signal sequence) of this chitinase was obtained by PCR cloning, and subsequently confirmed by immunological recognition of its overexpressed protein in Escherichia coli. Homology modeling predicted that this thermostable chitinase in jelly fig achenes comprised a stable (betaalpha)(8) barrel fold with three pairs of disulfide linkage. Immunostaining indicated that this chitinase was exclusively localized in the pericarpial region but not in the seed cells where bulky protein bodies and massive oil bodies were accumulated. Spore germination of Colletotrichum gloeosporioides, a common post-harvest pathogen infecting ripening fruit of jelly fig and many other fruits, was inhibited by this chitinase purified from achenes. It is suggested that the biological function of the thermostable chitinase in the pericarp of jelly fig achenes is to protect the nutritive seeds from fungal attack during fruit ripening.

N-acetyltransferase is involved in baicalein-induced N-acetylation of 2-aminofluorene and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells.

Many arylamine and hydrazine drugs are acetylated by cytosolic N-acetyltransferase (NAT). The human promyelocytic leukemia cell line (HL-60) has been shown to acetylate arylamine and contain NAT activity. The purpose of this study was to determine whether or not baicalein could affect N-acetylation of 2-aminofluorene (AF) in HL-60 cells. Acetylated and nonacetylated AF were determined by using high performance liquid chromatography. Baicalein displayed a dose-dependent inhibition of cytosolic and intact cells' NAT activity and reduced the number of viable cells. Time-course experiments showed that N-acetylation of AF, measured from intact HL-60 cells, was inhibited by baicalein for up to 48 h. Baicalein also decreased AF-DNA adduct formation in the examined cells. The effects of baicalein on NAT were examined by flow cytometry and NAT gene expression was examined by polymerase chain reaction. The results demonstrated that baicalein inhibited NAT1 mRNA gene expression and reduced the level of NAT in HL-60 cells. These results show that baicalein can affect the NAT activity of human leukemia cells in vitro.

Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes.

A method was developed to purify a 30-kDa protein from jelly fig (Ficus awkeotsang) pericarp, including preparation of jelly curd from achenes, extraction of proteins from the curd, and isolation of the 30-kDa protein by anion-exchanger and gel filtration. Chitinase activity was detected in the purified 30-kDa protein by activity staining in both non-denaturing gel electrophoresis and SDS-PAGE. Isoelectrofocusing showed that the isoelectric point of the 30-kDa protein was lower than pH 3.5. The K(m), k(cat), optimal pH and temperature of this putative chitinase were determined to be 0.076 mM, 0.089 s(-1), pH 4, and 60 degrees C, respectively. The purified 30-kDa protein was thermostable (retaining activity up to 65 degrees C for several hours) and could be stored at 4 degrees C for a year without apparent loss of chitinase activity. Antifungal activity of this putative chitinase was measured in terms of inhibition of Colletotrichum gloeosporioides spore germination.

Aloe-emodin inhibited N-acetylation and DNA adduct of 2-aminofluorene and arylamine N-acetyltransferase gene expression in mouse leukemia L 1210 cells.

N-Acetyltransferases (NATs) plays an important role in the first step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylatoion phenotypes, which are recognized to affect cancer risk related to environmental exposure. Aloe-emodin has been shown to exit anticancer activity. The purpose of this study is to examine whether or not aloe-emodin could affect arylamine N-acetyltransferase (NAT) activity and gene expression (NAT mRNA) and DNA-2-aminofluorene (DNA-AF) adduct formation in mouse leukemia cells (L 1210). By using high performance liquid chromatography, N-acetylation and non-N-acetylation of AF were determined and quantitated. By using reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, NAT mRNA was determined and quantitated. Aloe-emodin displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by aloe-emodin for up to 24h. Using standard steady-state kinetic analysis, it was demonstrated that aloe-emodin was a possible uncompetitive inhibitor to NAT activity in cytosols. The DNA-AF adduct formation in mouse leukemia cells were inhibited by aloe-emodin. The NAT1 mRNA in mouse leukemia cells were also inhibited by aloe-emodin. This report is the first demonstration which showed aloe-emodin affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and DNA-AF on adduct formation.

Oral administration of 5-methoxypsoralen affects the distribution and metabolism of 2-aminofluorene in Sprague-Dawley rats.

BACKGROUND: The effects of 5-methoxypsoralen (5-MOP) on the distribution and metabolism of chemical carcinogens such as 2-aminofluorene (AF) has not been previously reported. In this study, the influences of 5-MOP on the metabolism of AF in Sprague-Dawley (SD) rats were investigated. MATERIALS AND METHODS: After receiving 5-MOP in 24 hours, AF was introduced into each animal by gastric intubation. After 12, 24, 48 and 72 hours the urine, feces, and cytosol of the liver, kidneys, stomach, colon, bladder and blood of rats were collected and assayed for AF and its metabolites by HPLC. RESULTS: Compared to the control regimen, 5-MOP caused an increase of the metabolites excreted in urine and feces. The largest dose of metabolites were excreted between 48-72 hours. The major metabolite excreted in the urine was 9-hydroxy-AAF (9-OH-AAF) and in the feces was 7-hydroxy-AAF (7-OH-AAF). There was no time-effect for the tissues, and the liver was the main target organ for the AF and its metabolites. The major residual metabolite of AF in the liver, kidneys, stomach, colon and bladder was 7-OH-AAF. In blood it was 9-OH-AAF. The bladder had the lowest metabolic residue in tissues, and blood played the role of transportation but was not the target organ. 5-MOP decreased the concentration of AF and its residual metabolites of liver, stomach, kidneys, bladder and blood at various times. CONCLUSION: 5-MOP increased the metabolism of AF in order to transform to ring-hydroxylated metabolites and increased excretion of the ring-hydroxylated metabolites, therefore decreasing AF and its residual metabolites in vivo. Although 5-MOP was shown to be an inhibitor of CYP 2A6 and CYP 2B1, somehow it causes an increase of activity in AF metabolism in vivo; it induces more CYPs involved in the metabolism of AF.