scholarly journals Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors

1994 ◽  
Vol 300 (1) ◽  
pp. 117-124 ◽  
Author(s):  
L I McLellan ◽  
D J Judah ◽  
G E Neal ◽  
J D Hayes
Keyword(s):  
Rat Liver ◽  
Aflatoxin B1 ◽  
Resistance Mechanism ◽  
Constitutive Expression ◽  
Butylated Hydroxytoluene ◽  
Detoxification Enzymes ◽  
Rat Tissues ◽  
Aldehyde Reductase ◽  
Glutathione S Transferase ◽  
Adaptive Resistance

Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic content of AFB1-AR. Both BHA and BHT were also able to induce this enzyme but, by contrast, PB was found to be a poor inducer of AFB1-AR. AFB1, 3-MC and clofibrate were unable to serve as inducers of this reductase. The presence of Alpha-class GST, including the Yc2 subunit, was examined in various rat tissues. Constitutive expression of Yc2 was found in the epididymis at levels comparable with that observed in the liver from EQ-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues

2000 ◽  
Vol 348 (2) ◽  
pp. 389-400 ◽  
Author(s):  
Vincent P. KELLY ◽  
Linda S. IRELAND ◽  
Elizabeth M. ELLIS ◽  
John D. HAYES
Keyword(s):  
Rat Liver ◽  
Aflatoxin B1 ◽  
Single Gene ◽  
Reversed Phase ◽  
Cross Reactivity ◽  
Rat Tissues ◽  
Succinic Semialdehyde ◽  
Aldehyde Reductase ◽  
Cibacron Blue ◽  
Sds Page

Antiserum raised against human aflatoxin B1 aldehyde reductase 1 (hAFAR1) has been used to identify a previously unrecognized rat aldo-keto reductase (AKR). This novel enzyme is designated rat aflatoxin B1 aldehyde reductase 2 (rAFAR2) and it characteristically migrates faster during SDS/PAGE than does the archetypal ethoxyquin-inducible rAFAR protein (now called rAFAR1). Significantly, rAFAR2 is essentially unreactive with polyclonal antibodies raised against rAFAR1. Besides its distinct electrophoretic and immunochemical properties, rAFAR2 appears to be regulated differently from rAFAR1 as it is expressed in most rat tissues and does not appear to be induced by ethoxyquin. Multiple forms of rAFAR2 have been identified. Anion-exchange chromatography on Q-Sepharose, followed by adsorption chromatography on columns of Matrex Orange A and Cibacron Blue, have been employed to purify rAFAR2 from rat liver cytosol. The Q-Sepharose chromatography step resulted in the resolution of rAFAR2 into three peaks of AKR activity, two of which were purified and shown to be capable of catalysing the reduction of 2-carboxybenzaldehyde, succinic semialdehyde, 4-nitrobenzaldehyde and 9,10-phenathrenequinone. The two most highly purified rAFAR2-containing preparations eluted from the Cibacron Blue column were 91 and 98% homogeneous. Analysis of these by SDS/PAGE indicated that the least anionic (peak CBA5) comprised a polypeptide of 37.0 kDa, whereas the most anionic (peak CBA6) contained two closely migrating polypeptides of 36.8 and 37.0 kDa; by contrast, in the present study, rAFAR1 was estimated by SDS/PAGE to be composed of 38.0 kDa subunits. Final purification of the 37 kDa polypeptide in CBA5 and CBA6 was accomplished by reversed-phase HPLC. Partial proteolysis of the two preparations of the 37 kDa polypeptide with Staphylococcus aureus V8 protease yielded fragments of identical size, suggesting that they represent the product of a single gene. Furthermore, the peptide maps from CBA5 and CBA6 differed substantially from that yielded by rAFAR1, indicating that they are genetically distinct from the inducible reductase. A peptide generated by CNBr digestion of the 37 kDa polypeptide from CBA6 was shown by Edman degradation to share 88% sequence identity with residues Tyr168-Leu183 of rAFAR1. This provides evidence that the rat protein identified by its cross-reactivity with anti-hAFAR1 serum is an additional member of the AKR7 family.

scholarly journals Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide

1992 ◽  
Vol 285 (1) ◽  
pp. 173-180 ◽  
Author(s):  
J D Hayes ◽  
D J Judah ◽  
G E Neal ◽  
T Nguyen
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rat Liver ◽  
Cdna Clone ◽  
Aflatoxin B1 ◽  
Mouse Liver ◽  
High Catalytic Activity ◽  
Glutathione S Transferase ◽  
E Coli

Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to the constitutively expressed rat liver Yc1 subunit. The significance of the fact that both mouse Yc and rat Yc2 exhibit high catalytic activity towards AFB1-8,9-epoxide, whereas rat Yc1 possesses little activity towards this compound, is discussed in terms of structure/function.

scholarly journals A novel aldehyde reductase with activity towards a metabolite of aflatoxin B1 is expressed in rat liver during carcinogenesis and following the administration of an anti-oxidant

1993 ◽  
Vol 292 (1) ◽  
pp. 13-18 ◽  
Author(s):  
D J Judah ◽  
J D Hayes ◽  
J C Yang ◽  
L Y Lian ◽  
G C K Roberts ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Aflatoxin B1 ◽  
British Library ◽  
Aldehyde Reductase ◽  
Ph Values ◽  
Development Of Resistance ◽  
Neoplastic Lesions ◽  
Anti Oxidant ◽  
Rat Livers

In contrast with fractions from control animals, an aldehyde reductase, which catalyses the reduction of aflatoxin B1-dihydrodiol, in the dialdehyde form at physiological pH values, to aflatoxin B1-dialcohol, is expressed in cytosolic fractions prepared from rat livers bearing pre-neoplastic lesions, or following treatment with the anti-oxidant ethoxyquin. This expression parallels the development of resistance to the toxin. Unlike the aflatoxin B1-dihydrodiol, the dialcohol does not undergo binding to protein. This enzyme activity could play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Correlated n.m.r. and m.s. spectra are provided in Supplementary Publication SUP 50171 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

scholarly journals Substrate specificity of an aflatoxin-metabolizing aldehyde reductase

1995 ◽  
Vol 312 (2) ◽  
pp. 535-541 ◽  
Author(s):  
E M Ellis ◽  
J D Hayes
Keyword(s):  
Substrate Specificity ◽  
High Activity ◽  
Rat Liver ◽  
Aflatoxin B1 ◽  
Succinic Semialdehyde ◽  
Aldehyde Reductase ◽  
Aliphatic Aldehydes ◽  
Alpha Beta ◽  
Low Activity

The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactive with glucose, galactose and xylose. The enzyme also exhibits low activity towards alpha, beta-unsaturated carbonyl-containing compounds. Determination of the apparent Km reveals that AFAR has highest affinity for 9,10-phenanthrenequinone and succinic semialdehyde, and low affinity for glyoxal and DL-glyceraldehyde.

Glutathione-S-transferase, alcohol dehydrogenase and aldehyde reductase activities during diethylnitrosamine-carcinogenesis in rat liver

1993 ◽  
Vol 68 (2-3) ◽  
pp. 177-183 ◽  
Author(s):  
R.A. Canuto ◽  
G. Muzio ◽  
M. Maggiora ◽  
M.E. Biocca ◽  
M.U. Dianzani
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rat Liver ◽  
Aldehyde Reductase ◽  
Glutathione S Transferase
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