Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1

Moss, Elizabeth J.; Judah, David J.; Przybylski, Michael; Neal, G.E.

doi:10.1042/bj2100227

Cited by 53 publications

(26 citation statements)

References 24 publications

(21 reference statements)

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“…Another major metabolite detected was the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyl-AFB, (AFB,-N7-Gua) adduct, which accounted for 16% of the applied radioactivity. The level of AFB,-N7-Gua adduct in the urine agreed well with the amount calculated from the pharmacokinetic data of Moss et al (12). Overall recovery of the radioactive aflatoxins applied to the HPLC column was >95%.…”

Section: Resultssupporting

confidence: 73%

Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography.

Groopman¹,

Donahue²,

Zhu³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

149

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A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the afinity column. In studies on rats injected with ["'C]aflatoxin Bl, we identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFBl-N7-Gua), and the oxidative metabolites Ml and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins Ml and Pl. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.The aflatoxins are highly carcinogenic agents consistently found as contaminants in human food supplies in many areas of the world and epidemiologically linked to increased incidence of human liver cancer in Asia and Africa [see Busby and Wogan (ref. complex procedures used to purify the aflatoxins prior to analysis have seriously limited the application of these approaches in large-scale epidemiologic studies.A major objective of our work has been the development of rapid, noninvasive screening procedures for assessing the exposure of humans to environmentally occurring carcinogens. Useful protocols require the ability to quantify chemical carcinogens and their metabolites, especially DNA and protein adducts, in readily accessible compartments, such as urine and serum. We have been developing these monitoring techniques through immunoassays using monoclonal antibodies (8-10). These antibodies have proven to be useful analytical tools for quantifying the aflatoxins in biological fluids and also for use with affinity chromatography matrices as preparative tools to isolate aflatoxins from biological fluids (10). We report here results of initial applications of these techniques to the analysis of human urine obtained from people environmentally exposed to AFB1 in their diet and also of urine of rats injected with AFB1. These preparative and analytical procedures permit rapid measurement of aflatoxins in complex biological fluids under conditions that can be applied to large numbers of samples collected in epidemiologic surveys seeking to evaluate aflatoxin exposure as a risk factor for liver cancer in man.

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Section: Resultssupporting

confidence: 73%

Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography.

Groopman¹,

Donahue²,

Zhu³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

149

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“…Therefore the activity of GST toward the AFBO is a key factor in determining species resistance in some rodents. Several investigations on birds have reported an efficient CYP450 ability to bioactivate AFB1 (Neal et al, 1981(Neal et al, , 1986Klein et al, 2000Klein et al, , 2002 and a poor GST capacity to detoxify the toxin (Moss et al, 1983;Klein et al, 2000); however, the wide variations in sensitivity to AFB1 among poultry have not been fully explained through differences in AFB1 metabolism. It appears that several metabolic routes are involved in AFB1 biotransformation in poultry species, and their susceptibility to AFB1 cannot be explained as simply as in rats and mice.…”

Section: Discussionmentioning

confidence: 97%

“…It has been reported that rats have less GST activity toward AFBO than other rodents so there is a positive correlation between GST activity and AFB1 resistance (Monroe and Eaton, 1987;Hayes et al, 1998;Esaki and Kumagai, 2002). Although avian species are highly efficient in producing AFBO (Neal et al, 1981;Klein et al, 2000), they are not able to conjugate effectively the AFBO with GSH which indicates that they have low GST activity directed to this conjugation (Moss et al, 1983;Manson, 1995;Klein et al, 2000). Therefore, a mechanism that explains the wide variability of AFB1 sensitivity in poultry species has not been elucidated.…”

Section: Introductionmentioning

confidence: 95%

Microsomal and cytosolic biotransformation of aflatoxin B1 in four poultry species

Lozano

Diaz

2006

British Poultry Science

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1. This research evaluated differences in hepatic in vitro metabolism of aflatoxin B1 (AFB1) on selected avian species. 2. Microsomal and cytosolic liver fractions were obtained from chickens, ducks, quails and turkeys; eight males and eight females of each. 3. All microsomes studied produced AFB1-8,9-exo-epoxide (AFBO), a metabolite regarded as the active product of AFB1. Turkey microsomes produced 1.8 and 3.5 times more AFBO than quails and chickens microsomes, respectively. 4. Males from evaluated birds produced more AFBO than females, but statistically-significant differences between genders were observed only in ducks and turkeys. 5. The cytosolic fraction from all four species produced aflatoxicol (AFL). Turkey and duck hepatic cytosol produced more AFL than from quail and chickens. 6. It is known that turkeys are very sensitive to AFB1, quails are intermediate and chickens are particularly resistant; the differences in AFBO production shown in our study may help to explain the difference in vivo responses among turkeys, quail and chickens. 7. Moreover, AFL may be related to AFB1 toxicity; it was produced in larger amounts by hepatic cytosol from the more susceptible species. 8. Because AFBO production by microsomes in ducks was relatively low, it is possible that other toxicity mechanisms are involved in this highly susceptible species.

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“…The purified AFB-GSH coeluted from the HPLC column with enzymatically generated conjugate. The concentration of purified AFB-GSH was determined by measuring the absorbance at 365 nm using the extinction coefficient 21,800 M Ϫ1 (20). Glutathione S-transferase Expression and Purification-Escherichia coli BL21(DE3) were transformed with the pET11d plasmid containing the mGSTA3 cDNA and optimized for high levels of mGSTA3-3 protein expression.…”

Section: Methodsmentioning

confidence: 99%

Binding of the Aflatoxin-Glutathione Conjugate to Mouse Glutathione S-Transferase A3-3 Is Saturated at Only One Ligand per Dimer

McHugh

Atkins

Racha

et al. 1996

Journal of Biological Chemistry

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The binding of two different reaction products (p-nitrobenzyl glutathione and the aflatoxin-glutathione conjugate) to mouse glutathione S-transferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique. As expected, p-nitrobenzyl glutathione was found to bind with a stoichiometry of 2.24 ؎ 0.17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate, 8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B 1 (AFB-GSH), was found to bind with a stoichiometry of 1.12 ؎ 0.08 mol/mol of dimeric enzyme. p-Nitrobenzyl glutathione bound mGSTA3-3 with a dissociation constant (K d ) of 59 ؎ 17 M while the aflatoxinglutathione conjugate bound the enzyme with a K d of 0.86 ؎ 0.19 M. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a K i of 1.5 mM, suggesting that AFB-GSH was binding to the enzyme active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometry of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activity toward 1-chloro-2,4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene in the active site of the other subunit. To our knowledge, this is the first report of a glutathione S-transferase reaction product which binds to the enzyme with a stoichiometry of 1 mol/mol of dimer.Glutathione S-transferases (GSTs; EC 2.5. 1.18) 1 are a family of broad specificity detoxification enzymes which catalyze the conjugation of glutathione to xenobiotics and endogenous substrates with electrophilic functional groups. Conjugation of reactive substrates with glutathione by GST serves primarily to prevent deleterious reactions with nucleophilic centers in cellular macromolecules. As first shown by Benson et al. (1), overexpression of GST often provides protection against the effects of electrophile exposure. In contrast, depletion of glutathione can increase cellular damage caused by GST substrates (2). In vertebrates, the cytosolic GSTs consist of four classes of isoenzymes: alpha, mu, pi, and theta, which all have similar three-dimensional structures (3-6). The active form of GST is a dimer, either a homodimer or a heterodimer, composed of two subunits from the same class (7,8).Structural and functional studies have clearly shown that the GST homodimer has two identical binding sites (one per subunit) which act independently rather than cooperatively. Structures have been solved for numerous crystals of GST isoenzymes complexed with different GST products or product analogs in the active site. These crystals do not display any significant structural differences between the two subunits in the dimer, which might suggest differences in activity of the subunits (4, 5, 9, 10). In addition, many of these structures clearly show two products bound per dimer, one in the active site of each subunit (4, 5, 9 -11). Ligand binding experiments have shown noncooperative substrate and product binding with a stoichiometry of two ligands per dimer (12). In additio...

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Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1

Cited by 53 publications

References 24 publications

Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography.

Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography.

Microsomal and cytosolic biotransformation of aflatoxin B1 in four poultry species

Binding of the Aflatoxin-Glutathione Conjugate to Mouse Glutathione S-Transferase A3-3 Is Saturated at Only One Ligand per Dimer

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