Characterization of glutathione S-transferases in juvenile white sturgeon.

Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thus preventing toxicity. This study characterized the cytosolic GST classes of juvenile white sturgeon (Acipenser transmontanus) liver, using two methods of isolation. The first, which employed affinity chromatography, electrophoresis and immunoblotting against a polyclonal striped bass GST antibody, yielded two cytosolic GSTs. The GSTs were identified by nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS), peptide mass mapping and MS/MS sequencing, as well as de novo MS/MS sequencing as GST classes pi and mu using the Mascot search engine and the NCBI non-redundant database (nrDB) for both methods. The molecular masses were determined to be 23,548 +/- 23 and 26,027 +/- 23 Da, respectively, using linear matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The second method of isolation, which used affinity chromatography and high-pressure liquid chromatography (HPLC), yielded pi, mu, and possibly two alpha isoforms by MALDI-TOF-TOF, again searching against the NCBI nrDB. The alpha isoforms were determined to have molecular masses of 25,528 +/- 23 and 25,348 +/- 23 Da by electrospray ionization source (ESI)-MS. Overall, it appears that the HPLC method is more sensitive than immunoblotting with the current antibody. Activity of the cytosolic GSTs was evaluated using the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and found to be 2.4 +/- 0.6 U/mg cytosolic protein, and 0.41 +/- 0.05 U/mg cytosolic protein using ethacrynic acid (ETHA).

Direct liquid chromatography-mass spectrometry method for the detection of glutathione S-transferase isozymes and investigation of their expression in response to dietary flavone.

The cytosolic GSTs were measured directly in tissue homogenates using HPLC interfaced via ESI to a mass spectrometer (LC/MS). Total ion chromatograms were generated and filtered for ion currents corresponding to m/z ratio characteristic of individual GST isozymes. Direct LC/MS has a high degree of precision (8%) and low instrument detection limits (50-100 ng) and offers the advantage of monitoring GST expression at the protein level. In this study we describe the sub-chronic effect of feeding flavone (2500 mg/kg diet) on the expression of mGSTA3, mGSTP1, mGSTM1, and mGSTM2 in male and female mice. Additionally, we tentatively identify mGSTO and its up-regulation by flavone; a result that will facilitate the study of this novel enzyme. Flavone induced mGSTM1 and mGSTP1 in a gender and isozyme specific manner yet had no appreciable effect on the expression of mGSTA3. Male animals (day 5) displayed a 8-fold increase in mGSTM1 and a 2.6-fold increase in mGSTP1 whereas female animals displayed a 5- and 3-fold increase in mGSTM1 and mGSTP1, respectively. The mGSTM2 was detected only in flavone-fed animals, indicating an up-regulation of this isozyme by flavone. Results obtained using direct LC/MS compare favorably to the specific activity of individual isozymes (P = 0.19), and are comparable to GST levels determined using affinity chromatography followed by LC/MS (P = 0.79).

Isozyme- and gender-specific induction of glutathione S-transferases by flavonoids.

Most dietary flavonoids have antioxidant activity in vitro however, secondary mechanisms such as the ability to influence gene expression with the consequent modulation of specific enzymatic activities involved in the intracellular response against oxidative stress, are being realized. In the following study, we examined the ability of the flavonoids: flavone, morin, naringenin, (+)-catechin, and quercetin to modulate the activity of glutathione S-transferases (GSTs) mGSTA, mGSTP and mGSTM in hepatic tissues of male and female Swiss Webster mice. Subchronic dietary exposure to morin, naringenin, (+)-catechin, and quercetin (2,500 mg/kg diet for 20 days) did not produce statistically significant changes in GST activity. Conversely, gender-, and isozyme-specific induction of mGSTs were observed in animals fed flavone. A sevenfold increase in total mGST activity was observed in female animals whereas a fourfold increase was observed in male animals. Enzyme specific assays indicate that there were greater increases of both mGSTM (eightfold) and mGSTP (fourfold) activities in females as compared to males (sixfold and twofold, respectively). As testosterone is involved in the regulation of GSTs in mice, castrated males were fed flavone for 5 days (2,500 mg/kg diet). In this case, dietary flavone resulted in similar fourfold increases in total GST activity in inact and castrated animals. Isozyme specific studies indicate that increases could be attributed to an induction of mGSTM and mGSTP.

Effect of dietary constituents with chemopreventive potential on adduct formation of a low dose of the heterocyclic amines PhIP and IQ and phase II hepatic enzymes.

We conducted a study to evaluate dietary chemopreventive strategies to reduce genotoxic effects of the carcinogens 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). PhIP and IQ are heterocyclic amines (HCAs) that are found in cooked meat and may be risk factors for cancer. Typical chemoprevention studies have used carcinogen doses many thousand-fold higher than usual human daily intake. Therefore, we administered a low dose of [14C]PhIP and [3H]IQ and utilized accelerator mass spectrometry to quantify PhIP adducts in the liver, colon, prostate, and blood plasma and IQ adducts in the liver and blood plasma with high sensitivity. Diets supplemented with phenethylisothiocyanate (PEITC), genistein, chlorophyllin, or lycopene were evaluated for their ability to decrease adduct formation of [14C]PhIP and [3H]IQ in rats. We also examined the effect of treatments on the activity of the phase II detoxification enzymes glutathione S-transferase (GST), UDP-glucuronyltransferase (UGT), phenol sulfotransferase (SULT) and quinone reductase (QR). PEITC and chlorophyllin significantly decreased PhIP-DNA adduct levels in all tissues examined, which was reflected by similar changes in PhIP binding to albumin in the blood. In contrast, genistein and lycopene tended to increase PhIP adduct levels. The treatments did not significantly alter the level of IQ-DNA or -protein adducts in the liver. With the exception of lycopene, the treatments had some effect on the activity of one or more hepatic phase II detoxification enzymes. We conclude that PEITC and chlorophyllin are protective of PhIP-induced genotoxicity after a low exposure dose of carcinogen, possibly through modification of HCA metabolism.