Psoralen induces liver injury and affects hepatic bile acids metabolism in female and male C57BL/6J mice.

Psoralen is a major component of Fructus Psoraleae that could induce liver injury. In this study, C57BL/6J mice were administered with psoralen at doses of 80 mg/kg for 3, 7 and 14 days. Blood and liver samples were collected for serum biochemistry and histopathology examinations, respectively. Psoralen led to liver injury with significantly increased liver weight and liver coefficient and up regulated serum ALT, AST and TG but down regulated serum TC and TP. The expression of bile acid-associated transporters and enzymes was detected by western blot, and the results showed that psoralen significantly down-regulates the expressions of CYP7A1, CYP27A1, BSEP and OSTα protein while up-regulates the expressions of HMGCR and FASN, resulting in the obstacles of bile acid efflux in the liver. The contents of 24 kinds of bile acids in the liver were measured by LC-MS/MS, and the results showed that psoralen led to the accumulation of unconjugated bile acids in the liver, such as ALCA and CA, which were more severe in male mice than female mice. It was indicated that psoralen may disrupt the balance of bile acid metabolism by inhibiting the expression of the efflux transporter, which then leads to liver damage.

Psoralen induces hepatotoxicity by covalently binding to glutathione-S-transferases and the hepatic cytochrome P450.

BACKGROUND: Psoraleae Fructus has been widely used in China and its surroundings; however, Psoraleae Fructus and its compound preparation have been reported recently to cause liver injury in clinics. Thus, its safe use has attracted increasing attention. The possible mechanism is related to the metabolism of psoralen, but it still needs further clarification. PURPOSE: The present study was designed to evaluate the toxicity of psoralen and investigate the potentially related molecular mechanisms using chemical biology methods combined with animal experiments to provide evidence for the rational clinical use of psoralen. METHODS: An in vivo experiment was conducted with a time series of 20-80 mg/kg psoralen to verify its toxic performance. Target capture and click reactions were used to investigate direct targets of psoralen. Selectivity for different glutathione-S-transferase (GST) subtypes in the liver and inhibition of cytochrome P450 (CYP450) were also detected. RESULTS: Psoralen build-up in the liver is the primary cause of liver damage. Our study revealed the mechanism by which psoralen induces liver injury. Psoralen can bind directly to CYP2D6, CYP3A4, GST-α, and GST-µ and inhibit their activities, causing the depletion of glutathione (GSH) in vivo, which in turn induces hepatic damage. The special structure of α,ß-unsaturated lactones in psoralen facilitates its attachment to its target; therefore, complementing psoralen with GSH can efficiently protect the liver from damage. CONCLUSIONS: Psoralen causes a disorder in drug metabolism by inhibiting the activity of CYPs and GSTs, causing exhaustion of GSH, and subsequently leading to liver damage. The co-administration of GSH and psoralen is an effective way to avoid liver injury in clinical settings.

Effects of psoralen on hepatic bile acid transporters in rats.

Fructus Psoraleae (FP), widely used in traditional medicine, is increasingly reported to cause serious hepatotoxicity in recent years. However, the main toxic constituents responsible for hepatotoxicity and the underlying mechanisms are poorly understood. In the present study, psoralen, a main and quality-control constituent of FP, was intragastrically administered to Sprague-Dawley rats at a dose of 60 mg/kg for 1, 3 and 7 days. Blood and selected tissue samples were collected and analyzed for biochemistry and histopathology to evaluate hepatotoxicity. The results showed that psoralen could induce hepatotoxicity by enhanced liver-to-body weight ratio and alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total cholesterol after administration for 3 days. In addition, histopathological examinations also indicated the hepatotoxicity induced by psoralen. Furthermore, the mRNA and protein levels of hepatic bile acid transporters were significantly changed, in which MRP4, ABCG5 and ABCG8 were repressed, while the protein level of NTCP tended to increase in the rat liver. Taken together, psoralen caused liver injury possibly through affecting bile acid transporters, leading to the disorder of bile acid transport and accumulation in hepatocytes.

Transformation of Psoralen and Isopsoralen by Human Intestinal Microbial In Vitro, and the Biological Activities of Its Metabolites.

Psoralen (P) and isopsoralen (IP) are the main active ingredients in the dried fruit of Psoralen corylifolia L. (PC), with a wide range of pharmacology activities. The intestinal bacteria biotransformation plays a central role in the metabolism of the complex ingredients in traditional Chinese medicine (TCM). Our study aimed to investigated the metabolic profile of P and IP in the intestinal condition, co-cultured with human fecal bacteria anaerobically. Four bio-transforming products were obtained, including 6,7-furano-hydrocoumaric acid (P-1) and 6,7-furano-hydro- coumaric acid methyl ester (P-2), which transformed from P, and 5,6-furano-hydrocoumaric acid (IP-1) and 5,6-furano-hydrocoumaric acid methyl ester (IP-2), which were transformed from IP. It is worth mentioning that IP-2 is a new compound that has not been published. Their structures were analyzed based on their spectroscopic data. Moreover, a highly sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was used to characterize the metabolic pathways of P, IP, and their bio-transforming products in the reaction samples. In addition, the dampening effects against the oxidative stress of P, IP, and their bio-transforming products by human intestinal flora were estimated in vitro via the human colorectal cells (HCT116) and heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cell lines. The results showed that the metabolites have stronger activity than P and IP, which possibly provides a basis for elucidating the treating mechanisms of PC extract against inflammatory bowel disease.

Psoralen loaded liposomal nanocarriers for improved skin penetration and efficacy of topical PUVA in psoriasis.

Psoralen in combination with ultraviolet A radiation (PUVA) is an FDA recommended therapy for clinical application in the management of severe recalcitrant psoriasis. Psoralen acts by intercalation of DNA and upon exposure to UV-A, it forms monoadducts which in turn induce apoptosis. Poor skin deposition, weak percutaneous permeability of psoralen and adverse effects of severe burning, blisters, pigmentation associated with conventional topical psoralen vehicles hinders the therapeutic efficacy and safety of topical PUVA. The aim of the present study is to formulate psoralen loaded liposomal nanocarriers for enhanced skin penetration, safety and efficacy of topical PUVA in psoriasis. Two different liposomal compositions i.e., cationic liposomes composed of DC-Chol, cholesterol and anionic liposomes composed of egg lecithin, cholesterol, tetramyristoyl cardiolipin were prepared for the topical delivery of psoralen. Liposomal carriers were characterized with respect to size, zeta potential, entrapment efficiency, stability, in vitro drug release and in vivo studies. Both liposomes were prepared with particle size of nearly 100nm. Zeta potential and entrapment efficiency of cationic liposomes were +25.8mV, 75.12% and anionic liposomes were -28.5mV, 60.08% respectively. Liposomal dermal distribution demonstrated higher penetration of both liposomal carriers over solution. Similarly, skin permeation study indicated 5 fold increase in permeation of psoralen with liposomal carriers. Topical application of psoralen liposomal gels on imiquimod induced psoriatic plaque model reduced the symptoms of psoriasis and levels of key psoriatic cytokines such as tumor necrosis factor-α, IL-17 and IL-22. In conclusion, the developed liposomal carriers of psoralen were found to be promising and can find application for optimal safety and efficacy of topical PUVA in psoriasis.

In vitro transport mechanism of psoralen in a human placental cell line (BeWo cells).

The mechanism by which psoralen is transported across the placenta was investigated in the BeWo human placental cell line derived from choriocarcinoma in a transwell assay system using liquid chromatography-mass spectrometry/mass spectrometry detection. Psoralen uptake by BeWo cells increased linearly over the concentration range of 0.01 µM to 100 µM (r (2) = 0.997) and was not saturable. Psoralen uptake by BeWo cells was not affected by temperature (4 °C, room temperature, and 37 °C; p > 0.05). Psoralen transport increased linearly over 180 min (r (2) = 0.988) with 3.08 ± 0.26 %, 5.47 ± 0.21 %, 7.54 ± 0.06 %, 9.40 ± 0.37 %, 11.49 ± 0.31 %, and 12.46 ± 0.61 % transferred from the apical chamber to the basolateral chamber in the transwell assays at 30, 60, 90, 120, 150, and 180 min, respectively. The rate of transport showed the same tendency, increasing linearly from 0.13 ± 0.01 pmol/s to 0.58 ± 0.03 pmol/s over the concentration range of 25 µM to 100 µM (r (2) = 0.989). The apparent permeability coefficient for psoralen (100 µM) was 5.62 ± 0.24 × 10(-6) cm/s and 5.53 ± 0.47 × 10(-6) cm/s before and after treatment with verapamil (100 µM), respectively (p > 0.05). The efflux value for psoralen was approximately 1. These data show that psoralen is well absorbed and crosses the placental barrier via passive diffusion in the BeWo cell line.

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.

Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

Effect of polyamines on shoot multiplication and furanocoumarin production in Ruta graveolens cultures.

The influence of the polyamines putrescine (Put), spermine (Spr) and spermidine (Spd) on growth and furanocoumarin production was investigated by exogenous addition, at different concentrations, to shoot cultures of Ruta graveolens at different phases of growth. Preliminary studies indicated that addition of Put (20 microM) and Spr (80 microM) had a promotive effect on shoot multiplication rate and number of multiple shoots formed. Spd was toxic, even at lower concentrations. The growth-phase of the culture at the time of exogenous addition of polyamines was found to be an important factor. Put was most effective when added at the lag phase, while Spr was most effective when added in the log phase. Time course studies of growth and furanocoumarin content were carried out for each polyamine and phase of addition. It was seen that maximum production of furanocoumarins (256.8 mg/10 g DW) occurred in the second week when Put was added in the lag phase and 260.5 mg/10 g DW in the fourth week when Spr was added in the log phase. Put addition resulted in a 3.10 fold increase in psoralen, 6.12 in xanthotoxin and 1.46 fold in bergapten production. Spr addition resulted in a 1.31 fold increase in psoralen, 4.11 fold in xanthotoxin and 1.49 fold in bergapten production. Results indicate that alteration of growth and furanocoumarin production kinetics is a combined outcome of choice of polyamine and the phase of culture at the time of exogenous addition. Polyamine addition enabled significant enhancement in production of pharmaceutically important bergapten and xanthotoxin in shoot cultures of Ruta graveolens, which could be explored for commercial production.

The mitochondrial effects of novel apoptogenic molecules generated by psoralen photolysis as a crucial mechanism in PUVA therapy.

The generation of photoproducts of psoralen (POPs) might be relevant in cell death induced by psoralen plus UVA, namely PUVA, which is a recognized effective treatment for cutaneous T-cell lymphoma, chronic graft-versus-host disease, and psoriasis. We investigated the occurrence of POP-induced cell death and the underlying mechanisms. POPs were produced by irradiating a psoralen solution with UVA. Jurkat cells treated in the dark with these mixtures died mainly through an apoptotic mechanism. POPs were separated by high-performance liquid chromatography (HPLC), and cells were added with each of these fractions. A total of 2 dimers of psoralen and 6-formyl-7-hydroxycoumarin (FHC) were identified in the apoptogenic fractions. Apoptosis was preceded by mitochondrial dysfunction caused by the opening of the mitochondrial permeability transition pore (PTP). In fact, both mitochondrial depolarization and cell death were prevented by the PTP inhibitor cyclosporin A (CsA). PTP opening was also documented in isolated mitochondria added with POP, suggesting that apoptosis is caused by a direct effect of POP on mitochondria. In fact, FHC alone induced PTP opening and CsA-inhibitable cell death of Jurkat cells, whereas nontransformed T lymphocytes were resistant. Along with identifying novel apoptogenic molecules, the present results indicate that POP generation directs transformed cells to apoptosis.

Biotin-psolaren labeled cDNA amplicons for genotyping rotavirus strains by dot hybridization assay.

A simple method to produce biotinylated probes by utilizing an amine-psoralen to conjugate an ester-biotin to nucleic acid molecules is described. It is simple, rapid, and well suited to label cDNA probes with any PCR-generated amplicon for hybridization assay. Its application to identify or to confirm the G and P genotypes of rotavirus-derived amplicons is described; however, it may be used to label amplicons of any origin. As an alternative or as a complementary test to either PCR-typing assay and/or sequencing, it should reduce considerably the laboratory costs required to genotype fully virus strains in large epidemiological surveys conducted in developing countries.

Visualization of the annealing of complementary single-stranded DNA catalyzed by the herpes simplex virus type 1 ICP8 SSB/recombinase.

The rate of annealing of long linear complementary single-stranded (ss) DNAs can be increased greatly by certain DNA-binding proteins including the herpes simplex virus type 1 ICP8 SSB/recombinase. Using electron microscopy, we have investigated the DNA-protein structures involved in ICP8-mediated DNA annealing. We show that the formation of superhelical ICP8-ssDNA filaments is required for annealing. Two superhelices interact with each other to form a coiled-coil, which is the intermediate in annealing. In this process, the superhelices likely rotate and translocate relative to each other. Psoralen/UV photocrosslinking studies revealed that meta-stable contacts form at sites of limited sequence homology during the annealing. Partial proteolysis of ICP8 in the protein-ssDNA complexes showed that Mg2+ induces conformational changes in the N-terminal region (amino acid residues 1-305) of ICP8. In addition to Mg2+, Ca2+ and, to a significantly lesser extent, Cu2+ and Mn2+, were found to induce superhelix formation of the ICP8-ssDNA filament and to facilitate annealing. Mechanisms for how the coiled-coil structures facilitate annealing are discussed.

Reproducible and inexpensive probe preparation for oligonucleotide arrays.

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.