GERMAN / DEUTSCHES Morbus Meulengracht Forum > Allgemeines zu Morbus Meulengracht

Sulforaphan und UGT1a1

(1/6) > >>

()fenris:
http://carcin.oxfordjournals.org/cgi/content/full/23/8/1399




Fig. 1. Effect of sulforaphane on mRNA levels of UGT1A1 (solid square), GSTA1 (solid triangle) in HepG2 cells and UGT1A1 (solid circle) in HT29 cells. Cells were seeded at 4 x 104 (HepG2) or 15 x 104 (HT29) and exposed to 15 µM sulforaphane over a total of 18 h, against a DMSO control. Significant induction in HepG2 cells (P < 0.005) and in HT29 cells (P < 0.05) was observed from 0.5 to 18 h. Results are shown as mean and standard deviation (n > 3).




Fig. 2. Effect of sulforaphane concentration on induction of UGT1A1 (solid square) (P < 0.05, 15–30 µM), GSTA1 (solid triangle) (P < 0.01, 3–30 µM) in HepG2 cells, and UGT1A1 (solid circle) (P < 0.01, 3–30 µM) mRNA levels in HT29 cells. Cells were seeded at 4 x 104 (HepG2) or 15 x 104 (HT29) and treated at various concentrations (0.3–30 µM) of sulforaphane and incubated for 18 h. Fold induction against DMSO controls is plotted. No measurable cytotoxicity was observed even up to 30 µM or with the DMSO control.



Bilirubin glucuronidation and UGT1A1 protein expression
HepG2 and HT29 cells glucuronidated bilirubin; media alone yielded no conjugates. Treatment with sulforaphane resulted in a significant increase (P < 0.01) of excreted bilirubin glucuronides compared with DMSO controls (Table I). As expected, the basal activity of glucuronidation in HT29 cells was lower than that in HepG2 cells.



View this table:
[in this window]
[in a new window]
   Table I. Effect of sulforaphane treatment on bilirubin conjugation. HepG2 or HT29 cells were incubated with bilirubin (5 µM) in the presence of sulforaphane (15 µM) or DMSO control; values shown are the mean ± SD of three replicates with analysis by ANOVA. There was no detectable endogenous glucuronide formation in cell-free medium 
 

 
Figure 3 shows immunoblots using the UGT1A1 specific polyclonal antibody WB-UGT1A1. In control cells, almost no UGT1A1 protein can be detected. Treatment with sulforaphane produced a significant increase in UGT1A1 band intensity in both HepG2 and HT29 cells. These data confirm that the increased levels of glucuronidation (Table I) were at least partly due to increased UGT1A1 protein levels. These results demonstrate, for the first time, UGT1A1 protein levels and activity are inducible by sulforaphane in HT29 and HepG2 cells.


Induction of UGT1A1 and GSTA1 in HepG2 cells by sulforaphane-glutathione conjugate and sulforaphane nitrile
Figure 4 shows the effects of treating HepG2 cells with the alternative products of glucoraphanin, sulforaphane nitrile, and with a sulforaphane metabolite (glutathione conjugate). The latter induced GSTA1 with a similar potency to sulforaphane. When cells were treated with sulforaphane nitrile in addition to sulforaphane, no effect on induction of UGT1A1 compared to sulforaphane alone was observed. In contrast, the nitrile derivative did not induce significantly GSTA1 or UGT1A1.


Brassica vegetable consumption causes changes in cancer risk biomarkers, at least in part due to the content of isothiocyanates, derived from the parent glucosinolates (reviewed in ref. 32). Several reviews discuss the anti-carcinogenic properties of isothiocyanates (32–35), and this is at least in part through induction of phase II enzymes such as QR (15) and GST (16). For example, in humans, conjugation and excretion of the food-borne mutagen PhIP is increased after consumption of broccoli (36). Intact glucosinolates have low or no activity (37). Bioavailability of glucosinolates and isothiocyanates depend on the method of preparation of the vegetables (38) and on processing methods (39). Absorption of ITCs occurs in the small intestine in humans (38) and in rats (40) as well as from the colon (41), and isothiocyanates have been detected in plasma (38). ITC and nitriles are both found normally in broccoli and their amounts depend on storage and preparation conditions (42). As shown in Scheme 1, the isothiocyanate sulforaphane and sulforaphane nitrile are metabolites of the parent compound, 4-methylsulphinyl-butyl glucosinolate (glucoraphanin) (13,31). After formation of sulforaphane, but not the nitrile, conjugation with glutathione readily occurs both in vivo and in vitro. Metabolism of SFN to SFN-SG could occur in the plant (endogenous GSH) (43), in the gut lumen from GSH secreted in the bile (GSH 0.25 mM) (44), or intracellularly in the small intestine epithelium, in hepatic cells or in other cells (45). In rats and humans, the main product in urine of SFN and other ITC metabolism is N-acetyl cysteine, formed via a GSH conjugate (38,46). In the current study, we show that sulforaphane is a potent inducer of the glucuronidating enzyme, UGT1A1, at the levels of mRNA, protein and bilirubin activity in two human carcinoma cell lines, used as models for the liver (47) and colon (48). Conjugation with glutathione to form a dithiocarbamate does not significantly modify the activity of SFN. In marked contrast, sulforaphane nitrile does not induce either UGT1A1 or GSTA1. UGT1A1 activity is induced by sulforaphane in rodents, or cells derived from rodents: murine hepatoma cells (Hepa 1c1c7), male Wistar rats (49) and female Sprague–Dawley rats. UGT1A1 is inducible by other compounds such as chrysin in HepG2 (12,50) and HT29 cells (51). Chrysin is high affinity substrate for UGT1A1 with Km = 350 nM, and induced its own glucuronidation (12,52). It is interesting to note that SFN induced glucuronidation, but there is no evidence for glucuronidation of SFN.
Other activities have been reported for isothiocyanates. Sulforaphane stimulated extracellular signal regulated protein kinase 2 (erk2) but not JNK1 in HepG2 and Hepa1c1c7 cells, activated mitogen activated protein kinase (MAPK) and also stimulated raf kinase activity (53). SFN down-regulated COX-2 expression in Raw 264.7 macrophages at the transcriptional level but did not interact with nitric oxide directly and did not induce iNOS activity. SFN also selectively reduced binding of NF-B, but with no effect of LPS-induced degradation of I-B nor with nuclear translocation of NF-B (55). Isothiocyanates react readily with protein amino, sulfhydryl and tryptophan residues (56) and are taken up into cells by conjugation with glutathione (45). A two-fold induction of GST by sulforaphane in human hepatocytes has been reported (15), and sulforaphane may affect apoptosis in some systems (56). On the other hand, there is very little information on the biological activities of nitriles derived from glucosinolates. Nitriles derived from several glucosinolates are less effective at inhibiting cultured human K562 erythroleukemic cell growth than corresponding isothiocyanates (57). SFN nitrile was a poor inducer of quinone reductase in Hepa1c1c7 cells compared with SFN (14). The biological activities of dithiocarbamates have been reviewed, although not for glutathione conjugates of isothiocyanates from Brassica vegetables. Dithiocarbamates in general have numerous biological effects including increasing copper uptake into cells (58).

Induction of UGT1A1 increases conjugation of xenobiotics (9), which has the potential to reduce breast cancer risk (11) and of enhancing bilirubin clearance (8). A common genetic defect in the TATA box promoter of the UGT1A1 gene is associated with Gilbert’s syndrome causing mild hyperbilirubinaemia, and enhanced bilirubin clearance would be desirable in this condition. However, adverse effects of anticancer agents have been observed in Gilbert’s patients due to reduced drug or bilirubin glucuronidation (59) and so further human trials are needed to assess the role of drug–food interaction in a clinical environment. Decreased serum bilirubin levels have also been attributed to an increase in coronary heart risk, as bilirubin is believed to be an endogenous antioxidant preventing the formation of oxidised LDL and subsequent atherosclerosis (60). The results also suggest that sulforaphane treatment could be used to improve the potential of HepG2 cells as a model to study glucuronidation.

Medizinmann99:
Hallo,

bitte...hab jetzt eine Weile gebraucht bis ich die entsprechenden Breiten rausgefunden habe und dies jetzt global eingestellt d.h. man kann externe Bilder jetzt nicht mehr breiter machen als hier :)

Hm ok also für einen Nichtmathematiker wäre jetzt noch interessant, wie man das wohl am Besten allgemeinverständlich zusammenfassen kann...willst Du das versuchen oder soll ich?

()fenris:
ah ja hab mich schon gewundert, warum die bildadresse gleich geblieben ist.....
wir können es beide nochmal in unserem verständnis notieren...

auf jedn fall kann man die ugt1a1 auf das 2 fache pushen....
das ist schon mal gut....
bezogen auf menschliche Leberzellen, also kein rattenversuch...

Die frage ist nur wieviel sprossen wir dazu brauchen und wie oft man diese einnehmen muss.
Und wie das verhältnis von sulforophan und sulforophan nitrile bei sprossen ist.
Sie schreiben, dass Verhältnis auf zufall basiert....naja mal schaun...

()fenris:
To maximise you consumption of SF you need to eat lightly cooked broccoli florets. The light cooking is very important as it prevents nitrile formation. You need to leave some firmness in the floret to retain some myrosinase activity.

zitat professor

wir wissen, dass die myrosinase aktivität mit steigender Temperatur gegen null geht..

hinzu kommt laut prof. :
dass das leichte Kochen die nitrile formation verhindert.... somit entsteht vermehrt das bioaktive sulforaphan.

Somit muss man einen schwierigen Kompromiss eingehen...
einerseits erwärmen, damit die nitrile formation verhindert wird.....
andererseits darf es nicht zu heiss sein, sonst fehlt die myrosinase aktivität gegen null geht

die myrionase ist deshalb wichtig, da sie das sulf erst zum phase 2 stimulsator macht.
eine schwierige angelegenheit....

()fenris:
hier noch wichtige Textausschnitte.....

Myrosinase catalyzes the cleavage of glucose and the resulting intermediates rearrange to form one of several products, the most common of which are isothiocyanates and nitriles. Hydrolysis of the glucosinolate glucoraphanin, found in highest concentration in broccoli, releases the potent bioactive isothiocyanate sulforaphane or a relatively inactive sulforaphane nitrile. Crop development to enhance the health benefit of broccoli has focused on identification of broccoli varieties high in glucoraphanin.However, glucoraphanin hydrolysis in crushed broccoli favors nitrile formation, with as little as 5- 15 % of the glucoraphanin yielding bioactive sulforaphane. Processing technology is needed to optimize sulforaphane formation and enhance the health benefit from dietary broccoli.



Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 degrees C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 degrees C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.


An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-m cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties.

Navigation

[0] Themen-Index

[#] Nächste Seite