Use of the yeast Saccharomyces cerevisiae as a pre-screening approach for assessment of chemical-induced phototoxicity.

Photoreactive chemicals can induce dermatological reactions when present in the skin exposed to sunlight. Thus, new chemicals absorbing above 290 nm should have their potential phototoxicity tested. In order to screen a large number of molecules with various physico-chemical properties, a microbiological method is helpful. To this end, the yeast Saccharomyces cerevisiae was evaluated for its ability to detect phototoxic compounds. Twelve products known to be phototoxic in vivo and previously used as standards for validating the regulatory test 3T3 NRU were used in this work. Eleven of them could be detected in the yeast assay and, among them, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), angelicin and, to a lower extend, tiaprofenic acid induced genetic alterations. Interestingly, a pre-incubation with yeast cells in the dark before exposure decreased the phototoxicity of 5-MOP and 8-MOP but had no effect on this of chlorpromazine and ketoprofen. Saccharomyces cerevisiae and Salmonella typhimurium (strains TA100 and TA102) were compared for the evaluation of 5-MOP and 8-MOP photogenotoxicity; only the yeast assay allowed to perform experiments in exposure conditions close to those encountered in environmental situations. Finally, an application of this experimental approach to the detection of traces of furocoumarins in fragrance materials was developed.     

Disposition of 8-methoxypsoralen in the rat. Induction of metabolism in vivo and in vitro and identification of urinary metabolites by thermospray mass spectrometry

The pharmacokinetics and metabolism of 8-methoxypsoralen (8-MOP) were measured in the catheterized rat after pretreatment for 3 days with phenobarbital (PB), beta-naphthoflavone (BNF), 8-MOP, or vehicle. After an iv injection of 10 mg/kg of [14C]8-MOP, timed blood samples were collected and analyzed using a sensitive and specific assay for [14C]8-MOP. Total body clearance of 8-MOP increased from 0.55 +/- 0.06 liter/kg/hr in control rats to 5.6 +/- 0.4, 2.7 +/- 0.4, and 1.2 +/- 0.0 liters/kg/hr in rats pretreated with BNF, PB, and 8-MOP, respectively, indicating that all three compounds are inducers of 8-MOP metabolism. The pattern of urinary metabolites was altered by the enzyme inducers. The urinary excretion of the sulfate conjugate of 5-hydroxy-8-methoxypsoralen was increased from 10 to 40% of the dose after pretreatment with PB. This intact conjugate was identified using thermospray and fast atom bombardment mass spectrometry. Pretreatment with 8-MOP and BNF increased 2- and 3-fold, respectively, the urinary excretion of a labile sulfate conjugate of 5,8-dihydroxypsoralen. Metabolism of 8-MOP was demonstrated in the 9000 g supernatant and microsomes of rat liver and shown to be inducible by pretreatment of rats with BNF, PB, and 8-MOP. 8-MOP was metabolized in incubations with liver microsomes at rates of 0.22 +/- 0.06, 0.38 +/- 0.06, 0.78 +/- 0.07, and 0.91 +/- 0.03 nmol/min/mg of protein for vehicle, 8-MOP-, PB-, and BNF-pretreated rats, respectively. Results of our investigation indicate that the success of therapy with 8-MOP may be influenced by pharmacokinetic interactions with other drugs.     

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.     

Activation of 8-methoxypsoralen by cytochrome P-450. Enzyme kinetics of covalent binding and influence of inhibitors and inducers of drug metabolism

The kinetics of covalent binding of reactive metabolites of 8-methoxypsoralen (8-MOP) to protein were measured in incubations of liver microsomes of rats pretreated for 3 days with i.p. injections of 80 mg/kg/day of beta-naphthoflavone (BNF), phenobarbital (PB), 8-MOP, or vehicle. Covalent binding of radioactivity derived from [14C]8-MOP (labeled at the metabolically stable 4-position in the coumarin ring) required NADPH, obeyed classical Michaelis-Menten kinetics, and was inducible by both PB and BNF. Plots of V versus V/[S] were linear in liver microsomes of rats pretreated with vehicle, PB, or 8-MOP; respective values for Km were 26, 24 and 13 microM and for Vmax were 0.61, 1.70 and 0.50 nmol bound/min/mg protein. In microsomes of rats pretreated with BNF, high- and low-affinity components of covalent binding were observed with respective values for Km of 4.7 and 117 microM and for Vmax of 0.77 and 1.71 nmol bound/min/mg protein. Addition of glutathione and cysteine to the incubations decreased covalent binding by 33 and 67%, respectively, presumably by trapping reactive electrophilic metabolites. Inhibition of epoxide hydrolase with 1,1,1-trichloropropene-2,3-oxide did not affect covalent binding of reactive metabolites of 8-MOP. SKF-525A was a potent inhibitor of both the metabolism of 8-MOP and covalent binding in microsomes from rats pretreated with PB, but had only a slight effect in microsomes from rats pretreated with BNF. In contrast, alpha-naphthoflavone almost completely inhibited metabolism of 8-MOP and covalent binding in BNF-induced microsomes but had no effect in PB-induced microsomes. Apparent covalent binding was reduced by 39% in incubations with 8-MOP labeled with tritium in the metabolically labile methoxy group. Collectively, these results indicate that 8-MOP is biotransformed by two or more isozymes of cytochrome P-450 to reactive electrophiles capable of binding to tissue macromolecules.     

Inhibition and induction of drug metabolism by psoralens: alterations in duration of sleep induced by hexobarbital and in clearance of caffeine and hexobarbital in mice.

1. Hexobarbital (100 mg/kg i.p.) sleeping times in male CD-1 mice pretreated (-1 h) with a single i.p. injection of 150 mumol/kg of psoralen or coumarin analogues were increased, most markedly (6-fold) by linear, methoxy-substituted psoralens. 2. Hexobarbital sleeping times of mice which received three daily injections (231 mumol/kg; 50 mg/kg) of 8-methoxypsoralen (8-MOP) were 44% of controls (corn oil). 3. The whole-body half-life of caffeine (1 mg) in mice was 10.2, 1.2, and 0.37 h following 8-MOP (50 mg/kg per day) x 1, vehicle, and 8-MOP x 3 respectively. 4. The whole-body concentrations of hexobarbital (100 mg/kg dose) in mice 30 min after dosing were 14.3 +/- 0.9, 8.4 +/- 0.3, and 5.2 +/- 0.5 micrograms/ml (1 mouse = 150 ml) following 8-MOP (50 mg/kg per day) x 1, vehicle, and 8-MOP x 3 respectively. 5. It is concluded that, administered acutely, psoralen analogues inhibit hexobarbital metabolism in mice; and 8-MOP administered acutely inhibits the metabolism of caffeine and hexobarbital, but administered repeatedly increases their metabolism.     

Different modes of inhibition of mouse Cyp2a5 and rat CYP2A3 by the food-derived 8-methoxypsoralen.

CYP2A enzymes are responsible for nicotine metabolism and for activating tobacco-related carcinogens. Inhibition of CYP2A is a promising approach in chemoprevention, which could lead to a decrease in cigarette consumption and to a reduction in tobacco-related cancer risk. 8-Methoxypsoralen (8-MOP) is a mechanism-based inhibitor of human CYP2A6 and CYP2A13. 8-MOP is also an inhibitor of Cyp2a5, but the mode of this inhibition is unknown. There is no published data on the inhibition of CYP2A3 by 8-MOP. The objective of this work was to investigate the characteristics of 8-MOP inhibition on mouse hepatic Cyp2a5 and rat nasal CYP2A3, in order to determine the best experimental model for chemoprevention studies using 8-MOP. The results show that 8-MOP inhibits CYP2a5 through three different mechanisms: competitive, non-competitive (K(iu)=1.7 microM), and mechanism-based (K(inactivation) of 0.17 min(-1)). By contrast, 8-MOP was able to inhibit CYP2A3-mediated coumarin 7-hydroxylase only in a non-competitive way (K(iu)=0.22 microM). In conclusion, we showed that 8-MOP inhibits Cyp2a5 and CYP2A3 through different mechanisms.     

Inhibition and induction of drug metabolism by psoralens: Alterations in duration of sleep induced by hexobarbital and in clearance of caffeine and hexobarbital in mice

1. Hexobarbital (100?mg/kg i.p.) sleeping times in male CD-1 mice pretreated (-1?h) with a single i.p. injection of 150 μmol/kg of psoralen or coumarin analogues were increased, most markedly (6-fold) by linear, methoxy-substituted psoralens.

2. Hexobarbital sleeping times of mice which received three daily injections (231 μmol/kg; 50?mg/kg) of 8-methoxypsoralen (8-MOP) were 44% of controls (corn oil).

3. The whole-body half-life of caffeine (1?mg) in mice was 10˙2, 1˙2, and 0˙37?h following 8-MOP (50?mg/kg per day) × 1, vehicle, and 8-MOP × 3 respectively.

4. The whole-body concentrations of hexobarbital (100?mg/kg dose) in mice 30?min after dosing were 14˙3±0˙9, 8˙4±0˙3, and 5˙2±0˙5 μg/ml (1 mcuse = 150 ml) following 8-MOP (50?mg/kg per day) × 1, vehicle, and 8-MOP × 3 respectively.

5. It is concluded that, administered acutely, psoralen analogues inhibit hexobarbital metabolism in mice; and 8-MOP administered acutely inhibits the metabolism of caffeine and hexobarbital, but administered repeatedly increases their metabolism.     

不同组方祛白脂对小鼠B16黑素瘤细胞代谢的影响

目的：探寻祛白脂组方中影响小鼠B16黑素瘤细胞代谢的因素。方法：以小鼠B16细胞增殖活性、黑素生成量、酪氨酸酶活性为指标,以8-甲氧补骨脂素（8-MOP）浓度和辅药为考察因素,选用3×2析因设计研究不同组方祛白脂对小鼠B16黑素瘤细胞代谢的影响,用SPSSl 3.0软件辅助数据分析。结果：8-MOP 10μmol·L-1处理组小鼠B16黑素瘤细胞增殖能力明显高于其他组（P〈0.01）,8-MOP 50μmol·L-1处理组小鼠B16黑素瘤细胞黑素含量、酪氨酸酶活性明显高于其他组（P〈0.01）;加入辅药小鼠B16黑素瘤细胞增殖能力、黑素含量、酪氨酸酶活性显著高于不加辅药组（P〈0.01）;8-MOP浓度和辅药处理的交互作用对小鼠B16黑素瘤细胞增殖能力、黑素含量、酪氨酸酶活性有显著影响（P〈0.01）。结论：祛白脂中8-MOP浓度和辅药是影响小鼠B16黑素瘤细胞代谢的主要因素。     

Photochemotherapeutic Agent 8-Methoxypsoralen Induces Cytochrome P450 3A4 and Carboxylesterase HCE2: Evidence on an Involvement of the Pregnane X Receptor

8-Methoxypsoralen (8-MOP) is a prototype photochemotherapeuticagent and used to treat various skin disorders such as psoriasisand cutaneous T-cell lymphoma. Animal studies demonstrate thatrepeated treatment with 8-MOP markedly increases the capacityof drug metabolism. In this study, we report that 8-MOP is apotent inducer of cytochrome P450 3A4 (CYP3A4) and carboxylesterase2 (HCE2), two major human enzymes that catalyze oxidative andhydrolytic reactions, respectively. In human primary hepatocytes,8-MOP markedly induced the expression of CYP3A4 (approximatelysixfold) and HCE2 (approximately threefold) and the inductionoccurred in a concentration-dependent manner (0–50µM).RNA interference of the expression of the pregnane X receptor(PXR) proportionally decreased the induction. In a reporterassay, 8-MOP stimulated both CYP3A4 and HCE2 promoters, andthe stimulation was enhanced by cotransfection of PXR. Severalnatural variants of PXR differed markedly from the wild-typereceptor in responding to 8-MOP. In addition to human PXR (hPXR),8-MOP activated rat PXR, and the activation was comparable tothat of hPXR (EC₅₀ = 14µM). PXR is recognized as a masterregulator of the genes encoding drug-metabolizing enzymes andtransporters. The involvement of PXR in 8-MOP induction suggeststhat this chemotherapeutic agent causes a broader range of drug-druginteractions, and the differential activation of certain PXRvariants suggests that the magnitude of the interactions variesfrom person to person.     

Metabolism of the anti-psoriatic agent 5-methoxypsoralen in humans: comparison with rat and dog.

1. Single oral doses of 14C-5-methoxypsoralen (5-MOP) to human subjects (50 mg), rats (1 mg/kg) and dogs (1 mg/kg) were fairly well absorbed but subjected to extensive first-pass metabolism, at least in rat and human. Means of 62, 51 and 40% dose in urine and 31, 38 and 48% dose in faeces, were excreted by humans (during 5 days), rats (3 days) and dogs (1 day), respectively. In dogs, faecal 14C was probably derived, in part, from biliary excreted material. 2. Total 14C in human plasma reached peak concentrations after 2 h (mean 235 ng 5-MOP equivalent/ml) and declined relatively slowly, to about 60% of this value within 24 h. Unchanged 5-MOP was not detected in plasma using h.p.l.c. (< 5 ng/ml). 3. Tissue concentrations of 14C were generally greater in dogs than rats and reached peak levels at 1 h in dogs but at 24 h in rats. Apart from liver and bile, dog tissue 14C concentrations were lower than those in the corresponding plasma, whereas in rat they were lower only until the time of peak concentrations, after which they were generally greater. 4. 5-MOP was extensively metabolized in all three species. The major 14C-components in human and dog urine were glucuronic acid conjugates, mainly of an arylacetic acid and arylalcohols, resulting from initial oxidative metabolism of the furan ring of 5-MOP. In rat, these metabolites were excreted mainly unconjugated. An unusual metabolite was formed by reduction of the lactone moiety of 5-MOP, probably by the gut flora, giving rise to an arylpropionic acid, excreted as a glucuronic acid conjugate in the urine of all three species. 5. Unchanged drug was a very minor component of human and rat plasma, but a major component of dog plasma. In all three species, circulating 14C-metabolites were similar to those in the urine but were present mainly unconjugated. On the basis of these data, the metabolic fate of 5-MOP in humans was more similar to that in dog than to that in rat, although humans appeared to metabolize 5-MOP more rapidly than did dog.     

Inhibition and induction of theophylline metabolism by 8-methoxypsoralen. In vivo study in rats and humans

The effects of 8-methoxypsoralen (8-MOP) on the metabolism of theophylline were studied in rats and humans. Rats were randomized into three groups and prepared with iv jugular catheters. Group I (N = 4) received a single ip injection of 27 mg/kg of 8-MOP, group II (N = 5) vehicle (corn oil), and group III (N = 4) 50 mg/kg/day of 8-MOP for 3 days. Rats were subsequently administered 15 mg/kg of theophylline iv, and timed blood samples (0.2 ml) were assayed for theophylline by HPLC. Theophylline clearance (ml/min/kg; mean +/- SD) was 1.7 +/- 0.3, 2.4 +/- 0.5, and 9.5 +/- 1.6 in groups I, II, and III, respectively. The half-life (harmonic mean) from 0.5 to 12 hr was 7.2, 3.6, and 0.8 hr. Urinary excretion of unchanged theophylline (mean +/- SD) from 0 to 24 hr was 60 +/- 10, 41 +/- 6, and 13 +/- 3% of the administered dose. In a crossover study, three healthy, male, nonsmokers received 600 mg of oral theophylline. Urine and plasma were collected for 48 hr. One week later, subjects received 1.2 mg/kg of oral 8-MOP followed in 1 hr by 600 mg of oral theophylline. Mean residence time of theophylline increased from 10.7, 17.2, and 12.2 hr in the control period, to 20.3, 19.0, and 18.4 hr after 8-MOP. The AUC (microgram.hr/ml) of theophylline increased from 204, 213, and 204, to 555, 364, and 432, while clearance (ml/min/kg) decreased from 0.74, 0.57 and 0.63, to 0.27, 0.33, and 0.30, respectively. Urinary excretion of unchanged theophylline from 0 to 48 hr increased from 14, 14, and 15, to 24, 21, and 20%. We conclude that 8-MOP administered acutely is a potent inhibitor of theophylline metabolism and chronically in the rat is a powerful inducer.     

Effect of polyamidoamine dendrimer G3 and G4 on skin permeation of 8-methoxypsoralene--in vivo study

In the present study we have assessed the ability of (PAMAM) dendrimers G3 and G4 to facilitate transdermal delivery of 8-methoxypsoralen (8-MOP) in vivo. In vitro study using Franz diffusion cell revealed an enhanced transdermal flux for 8-MOP in complex with G3 and G4 dendrimer in relation to standard 8-MOP solution. In present study in vivo skin permeation potential of 8-MOP complex with G3 and G4 PAMAM dendrimer was assessed using confocal laser scanning microscopy (CLSM), which revealed an enhanced permeation of the 8-MOP to the deeper layers of the skin and significantly higher concentration in comparison with standard 8-MOP solution. Skin tissue 8-MOP concentration, evaluated by HPLC indicates that G3 and G4 PAMAM application significantly increase 8-MOP skin deposition in comparison with standard 8-MOP solutions after 1 and 2h. G4 appeared to be a more effective 8-MOP penetration enhancer than G3 PAMAM. Our results suggest the feasibility of G3 and G4 PAMAM dendrimers for transdermal delivery of 8-MOP resulting in better skin permeation and higher concentration of 8-MOP in epidermis and dermis of the drug that could help to improve effectiveness and safety of PUVA therapy.     

Apiaceous vegetable constituents inhibit human cytochrome P-450 1A2 (hCYP1A2) activity and hCYP1A2-mediated mutagenicity of aflatoxin B1.

In humans, apiaceous vegetables (carrots, parsnips, celery, parsley, etc.) inhibit cytochrome P-450 1A2, a biotransformation enzyme known to activate several procarcinogens, including aflatoxin B1 (AFB). We evaluated eight phytochemicals from apiaceous vegetables for effects on human cytochrome P-450 1A2 (hCYP1A2) activity using a methoxyresorufin O-demethylase (MROD) assay and a trp-recombination assay. Saccharomyces cerevisiae was used for heterologous CYP1A2 expression and this yeast strain is also diploid and auxotrophic for tryptophan due to mutations in the trp5 alleles. When these two alleles undergo AFB-induced mitotic recombination, gene conversion occurs, allowing yeast to grow in the absence of tryptophan. The apiaceous constituents psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and apigenin were potent inhibitors of hCYP1A2-mediated MROD activity in yeast microsomes, whereas quercetin was a modest hCYP1A2 inhibitor. Naringenin, caffeic acid, and chlorogenic acid did not inhibit hCYP1A2-mediated MROD activity. The 2-h pretreatment of intact yeast cells with psoralen, 5-MOP, and 8-MOP significantly improved cell survival after subsequent 4-h AFB treatment and reduced hCYP1A2-mediated mutagenicity of AFB. Apigenin also significantly decreased mutagenicity. These results suggest that in vivo CYP1A2 inhibition by apiaceous vegetables may be due to the phytochemicals present and imply that apiaceous vegetable intake may be chemopreventive by inhibiting CYP1A2-mediated carcinogen activation.     

8-Methoxypsoralen Induces Apoptosis by Upregulating p53 and Inhibits Metastasis by Downregulating MMP-2 and MMP-9 in Human Gastric Cancer Cells

Furanocoumarin 8-methoxypsoralen (8-MOP) is the parent compound that naturally occurs in traditional medicinal plants used historically. 8-MOP has been employed as a photochemotherapeutic component of Psoralen + Ultraviolet A (PUVA) therapy for the treatment of vitiligo and psoriasis. Although the role of 8-MOP in PUVA therapy has been studied, little is known about the effects of 8-MOP alone on human gastric cancer cells. In this study, we observed anti-proliferative effect of 8-MOP in several human cancer cell lines. Among these, the human gastric cancer cell line SNU1 is the most sensitive to 8-MOP. 8-MOP treated SNU1 cells showed G1-arrest by upregulating p53 and apoptosis by activating caspase-3 in a dose-dependent manner, which was confirmed by loss-of-function analysis through the knockdown of p53-siRNA and inhibition of apoptosis by Z-VAD-FMK. Moreover, 8-MOP-induced apoptosis is not associated with autophagy or necrosis. The signaling pathway responsible for the effect of 8-MOP on SNU1 cells was confirmed to be related to phosphorylated PI3K, ERK2, and STAT3. In contrast, 8-MOP treatment decreased the expression of the typical metastasis-related proteins MMP-2, MMP-9, and Snail in a p53-independent manner. In accordance with the serendipitous findings, treatment with 8-MOP decreased the wound healing, migration, and invasion ability of cells in a dose-dependent manner. In addition, combination treatment with 8-MOP and gemcitabine was effective at the lowest concentrations. Overall, our findings indicate that oral 8-MOP has the potential to treat early human gastric cancer, with fewer side effects.     

Metabolism of the anti-psoriatic agent 5-methoxypsoralen in humans: comparison with rat and dog

1. Single oral doses of ₁₄C-5-methoxypsoralen (5-MOP) to human subjects (50 mg), rats (1 mg/kg) and dogs (1 mg/kg) were fairly well absorbed but subjected to extensive first-pass metabolism, at least in rat and human. Means of 62, 51 and 40% dose in urine and 31, 38 and 48% dose in faeces, were excreted by humans (during 5 days), rats (3 days) and dogs (1 day), respectively. In dogs, faecal ₁₄C was probably derived, in part, from biliary excreted material.

2. Total ₁₄C in human plasma reached peak concentrations after 2 h (mean 235 ng 5-MOP equivalent/ml) and declined relatively slowly, to about 60% of this value within 24 h. Unchanged 5-MOP was not detected in plasma using h.p.l.c. (< 5 ng/ml).

3. Tissue concentrations of ₁₄C were generally greater in dogs than rats and reached peak levels at 1 h in dogs but at 24 h in rats. Apart from liver and bile, dog tissue ₁₄C concentrations were lower than those in the corresponding plasma, whereas in rat they were lower only until the time of peak concentrations, after which they were generally greater.

4. 5-MOP was extensively metabolized in all three species. The major ₁₄C-components in human and dog urine were glucuronic acid conjugates, mainly of an arylacetic acid and arylalcohols, resulting from initial oxidative metabolism of the furan ring of 5-MOP. In rat, these metabolites were excreted mainly unconjugated. An unusual metabolite was formed by reduction of the lactone moiety of 5-MOP, probably by the gut flora, giving rise to an arylpropionic acid, excreted as a glucuronic acid conjugate in the urine of all three species.

5. Unchanged drug was a very minor component of human and rat plasma, but a major component of dog plasma. In all three species, circulating ₁₄C-metabolites were similar to those in the urine but were present mainly unconjugated. On the basis of these data, the metabolic fate of 5-MOP in humans was more similar to that in dog than to that in rat, although humans appeared to metabolize 5-MOP more rapidly than did dog.     

Dark effect of 8-methoxypsoralen on human erythrocytes

Abstract— Furanocoumarin 8-methoxypsoralen (8-MOP) (1–100 μg mL^?1) in the dark showed a protective affect against hypotonic haemolysis of the erythrocyte membrane. However, the effect against heat-induced haemolysis was dependent on the concentration of 8-MOP; lower concentrations of 8-MOP showed an inhibiting effect, whereas higher concentrations caused acceleration of haemolysis. 8-MOP was not able to induce haemolysis in isotonic solution at 20 or 37 C. Reaction of erythrocytes with 8-MOP in the dark resulted in a shrinkage of the cells and alterations of their shapes. We conclude that modification of erythrocyte membrane by 8-MOP proceeds via reaction with membrane lipids and proteins. This indicates that the effect on the cell membrane plays an important role in the mechanism of the action of 8-MOP on the cells.     

Down regulation of differentiated embryonic chondrocytes 1 (DEC1) is involved in 8-methoxypsoralen-induced apoptosis in HepG2 cells

8-Methoxypsoralen (8-MOP), a naturally occurring compound, is a potent modulator of epidermal cell growth and differentiation in combination with ultraviolet light. However, there is little information on 8-MOP contribution to cell apoptosis alone. In the study, we evaluated 8-MOP, independently of its photoactivation, induced apoptosis in human hepatocellular carcinoma HepG2 cells. And we provide a molecular explanation linking 8-MOP to induce apoptosis. In HepG2 cells, treatment with 8-MOP induced the cell apoptosis in both dose-dependent and time-dependent manners. IC(50) values of 8-MOP were 8.775, 5.398μM for 48 and 72h, respectively. Further study showed that 8-MOP decreased the procaspase-3, procaspase-8, and procaspase-9, increased the ratio of Bax/Bcl-2 and decreased the survivin. Moreover, 8-MOP decreased differentiated embryonic chondrocyte gene1 (DEC1). Overexpression of DEC1 antagonized partially apoptosis induced by 8-MOP. And overexpression of DEC1 abolished the decrease of survivin and the activation of caspase-3 induced by 8-MOP partially. So, down regulation of DEC1 is involved in 8-MOP-induced apoptosis in HepG2 cells. Here, it is demonstrated that DEC1 possesses anti-apoptotic effects in 8-MOP-treated HepG2 cells. The findings provide more of a basis for 8-MOP as an anti-tumor agent in cancer therapy.     

Effects of 5-methoxypsoralen (5-MOP) on arylamine N-acetyltransferase activity in the stomach and colon of rats and human stomach and colon tumor cell lines

BACKGROUND: It has been shown that cytochrome P450 enzymes (CYPs) and acetyltransferase can be used as biomarkers of carcinogen-DNA adduct levels and human cancer susceptibility. The gastrointestinal tract is the portal of entry of foreign compounds and presents xenobiotic metabolizing N-acetyltransferase (NAT) and CYPs activities. 5-Methoxypsoralen (5-MOP) has been used in combination with UV radiation in skin photochemotherapy for decades. A number of studies have demonstrated that 5-MOP is inhibitory towards mouse and human CYP isoforms, but investigations on the direct effects on NAT activity in laboratory animals and human cancer cells are limited. The main objective of this study was to document the effects of 5-MOP on the modulation of NAT activities in the stomach and colon of rats and human stomach and colon tumor cell lines. MATERIALS AND METHODS: N-Acetylation of 2-aminofluorene (AF) to 2-acetylaminofluorene (AAF) by NAT in the stomach and colon of Sprague-Dawley (SD) rats and in human stomach (SC-M1) and colon (COLO 205) tumor cell lines was investigated. RESULTS: The data show that the metabolic activity of NAT in the rat colon was higher than that in the rat stomach, and the further metabolism of AAF was slower in the stomach than in the colon. 5-MOP increased the activity of NATand also increased the further metabolism of AAF at 24 h in the rat stomach. In the rat colon, no statistically significant changes caused by 5-MOP were observed in NAT activity, but 5-MOP increased the further metabolism of AAF at 24 to 72 h. 5-MOP decreased the activity of NAT only at 72-h incubation in SC-M1 cells. In COLO 205 cells, however, 5-MOP decreased the activity of NAT between 24 h and 72 h. The optimal concentrations of 5-MOP to induce decreased NAT activity in SC-M1 cells were 0.05 mM to 25 mM. In COLO 205 cells, the data indicate that the higher the concentrations of 5-MOP, the higher the acetylation of AF; a promotion effect of NAT activity occured at a higher dose (50 mM) of 5-MOP and an inhibition effect occured at lower doses (0.05-0.5 mM) of 5-MOP, while concentrations of 5-25 mM of 5-MOP showed no significant difference compared with the control regimen. CONCLUSION: The metabolic activity of NAT in the rat colon was higher than that in the rat stomach, and the results also showed a high degree of correspondence with SC-M1 cells and COLO 205 cells. 5-MOP more efficiently inhibited NAT activity in human stomach and colon tumor cell lines than in the stomach and colon of rats.     

8-甲氧补骨脂素脂质体凝胶对白癜风模型豚鼠的治疗作用研究

目的 :比较8 -甲氧补骨脂素 (8 -MOP)脂质体凝胶与其等浓度的其它剂型对白癜风模型豚鼠的疗效。方法 :将豚鼠分为正常组、模型组、8 -MOP酊剂组、8 -MOP凝胶组和8 -MOP脂质体凝胶组。除正常组外 ,其余各组均用化学脱色法造模 ,并给以相应药物治疗 ,之后比较各组疗效 ,同时考察各组表皮毛囊黑色素生成、血清胆碱酯酶及皮肤丙二醛 (MDA )含量等指标的变化。结果 :与其它各组比较 ,8 -MOP脂质体凝胶组总有效率更高 ,该组的毛囊黑色素生成、血清胆碱酯酶及MDA含量等指标 ,与模型组比较具有极显著性差异 (P<0.001) ,与凝胶组及酊剂组比较具有显著性差异 (P<0.05)。结论 :8 -MOP脂质体凝胶较其酊剂和凝胶对白癜风模型豚鼠具有更好的治疗作用。     

Dietary intake and risk assessment of phototoxic furocoumarins in humans

The question of whether the furocoumarin content of vegetables is sufficient to induce phototoxic skin reactions after ultraviolet irradiation was examined in two experiments with four human volunteers. In a first experiment, 300 g of celery roots (total phototoxic furocoumarin content 28.2 micrograms/g) was ingested. No skin reactions were observed after UVA exposure (1.5-9 J/cm2), and the blood levels of the furocoumarins--psoralen, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP)--were below the analytical detection limit of 2 ng/ml. To investigate the phototoxic effects of a mixture of the two most important furocoumarins in vegetables, after gastro-intestinal uptake, 8-MOP and 5-MOP (15 mg each) were ingested separately in a 50% alcoholic solution. A strong and persistent erythema was induced in three out of the four subjects (UVA dose: 3-25 J/cm2). The blood levels immediately before UVA irradiation varied between 14 and 114 ng/ml (8-MOP), and 17 and 70 ng/ml (5-MOP). In the subject who did not show phototoxicity, the blood levels remained at trace levels (3 ng/ml). Two subjects were also tested with a mixture of 10 mg 8-MOP plus 10 mg 5-MOP; in one subject the mixture induced pigmentation only, while in the other a mild-to-medium erythema was induced. The blood levels of the furocoumarins in the two subjects were similar (12-15 ng/ml for 8-MOP and 5-MOP). It is concluded that in humans the phototoxic threshold dose of furocoumarin mixtures is of the order of 10 mg 8-MOP plus 10 mg 5-MOP, which is equivalent to about 15 mg 8-MOP per person (blood levels of 8-MOP and 5-MOP at 30 min after ingestion of about 10-15 ng/ml each). This phototoxic threshold dose was not reached by the consumption of celery roots and other conventional vegetables under normal dietary habits (experimental intake of 2-8 mg per subject of the potentially phototoxic furocoumarin mixture). However, the safety factor between the possible actual intake of furocoumarins and the phototoxic threshold dose is about 2-10, which is relatively small.