Limits...
Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals

View Article: PubMed Central - PubMed

ABSTRACT

Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans.

No MeSH data available.


e-GST and pseudo-oxidized e-GST in bovine blood. (a) Mean level of e-GST in: total bovine blood, blood treated with the reducing agent DTT, isolated erythrocytes, isolated erythrocytes treated with DTT, bovine serum, bovine serum treated with DTT, bovine serum treated with DTT and incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration), and serum treated with DTT and assayed without the substrate GSH. The last two columns on the right report the reaction of purified BSA (in the same assay concentration of serum samples, 2.3 μM) with CDNB (1 mM, pH 6.5) before and after reduction with DTT. (b) Recombinant purified GSTP1-1, GSTA1-1 and GSTM2-2 were added to bovine serum after DTT reduction to reach the reported activities. Each samples were then incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration). Last column on the right shows the activity in serum treated with DTT and filtered by Ultracel cutoff 10 kDa (Amicon, Merck Millipore, Darmstadt, Germany). Error bars are the S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4979413&req=5

fig2: e-GST and pseudo-oxidized e-GST in bovine blood. (a) Mean level of e-GST in: total bovine blood, blood treated with the reducing agent DTT, isolated erythrocytes, isolated erythrocytes treated with DTT, bovine serum, bovine serum treated with DTT, bovine serum treated with DTT and incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration), and serum treated with DTT and assayed without the substrate GSH. The last two columns on the right report the reaction of purified BSA (in the same assay concentration of serum samples, 2.3 μM) with CDNB (1 mM, pH 6.5) before and after reduction with DTT. (b) Recombinant purified GSTP1-1, GSTA1-1 and GSTM2-2 were added to bovine serum after DTT reduction to reach the reported activities. Each samples were then incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration). Last column on the right shows the activity in serum treated with DTT and filtered by Ultracel cutoff 10 kDa (Amicon, Merck Millipore, Darmstadt, Germany). Error bars are the S.E.M.

Mentions: As mentioned above, the human GSTP1-1 may form an oxidized enzyme involving the two more reactive cysteines (i.e., Cys47 and Cys101).9 The resulting oxidized enzyme is fully inactive.9 The presence of oxidized GSTP1-1 has recently been discovered in human saliva,11 but it has never been observed in mammalian blood. After incubation of hemolyzed total blood with DTT, we observed a conjugation rate of GSH with CDNB higher than that found in the absence of reducing treatment, suggesting the presence of a significant, not spurious amount of oxidized e-GST (Figure 2a). Surprisingly, this additional activity was not recovered in isolated erythrocytes, but only in serum (Figure 2a). In a recent reinvestigation of the possible presence of GSTP1-1 in human serum, we concluded that it was absent or below the detection limit of the usual spectrophotometric assay,12 but we did not verify the possible occurrence of oxidized GSTP1-1. So far, no data are available in the literature concerning the presence of oxidized GSTP1-1 in bovine blood. Further experiments to clarify this point gave surprising results. In fact, the addition of NBDHEX, a strong and specific inhibitor of e-GST and other GST isoenzymes belonging to the alpha, pi and mu classes,13 did not suppress this additional GST activity (Figure 2b), and curiously the activity was also found in the absence of GSH (Figure 2a). Thus, the additional activity observed after reduction with DTT cannot be explained by the presence of oxidized GST in the samples.


Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals
e-GST and pseudo-oxidized e-GST in bovine blood. (a) Mean level of e-GST in: total bovine blood, blood treated with the reducing agent DTT, isolated erythrocytes, isolated erythrocytes treated with DTT, bovine serum, bovine serum treated with DTT, bovine serum treated with DTT and incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration), and serum treated with DTT and assayed without the substrate GSH. The last two columns on the right report the reaction of purified BSA (in the same assay concentration of serum samples, 2.3 μM) with CDNB (1 mM, pH 6.5) before and after reduction with DTT. (b) Recombinant purified GSTP1-1, GSTA1-1 and GSTM2-2 were added to bovine serum after DTT reduction to reach the reported activities. Each samples were then incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration). Last column on the right shows the activity in serum treated with DTT and filtered by Ultracel cutoff 10 kDa (Amicon, Merck Millipore, Darmstadt, Germany). Error bars are the S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979413&req=5

fig2: e-GST and pseudo-oxidized e-GST in bovine blood. (a) Mean level of e-GST in: total bovine blood, blood treated with the reducing agent DTT, isolated erythrocytes, isolated erythrocytes treated with DTT, bovine serum, bovine serum treated with DTT, bovine serum treated with DTT and incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration), and serum treated with DTT and assayed without the substrate GSH. The last two columns on the right report the reaction of purified BSA (in the same assay concentration of serum samples, 2.3 μM) with CDNB (1 mM, pH 6.5) before and after reduction with DTT. (b) Recombinant purified GSTP1-1, GSTA1-1 and GSTM2-2 were added to bovine serum after DTT reduction to reach the reported activities. Each samples were then incubated with the specific GST inhibitor NBDHEX (0.1 mM final concentration). Last column on the right shows the activity in serum treated with DTT and filtered by Ultracel cutoff 10 kDa (Amicon, Merck Millipore, Darmstadt, Germany). Error bars are the S.E.M.
Mentions: As mentioned above, the human GSTP1-1 may form an oxidized enzyme involving the two more reactive cysteines (i.e., Cys47 and Cys101).9 The resulting oxidized enzyme is fully inactive.9 The presence of oxidized GSTP1-1 has recently been discovered in human saliva,11 but it has never been observed in mammalian blood. After incubation of hemolyzed total blood with DTT, we observed a conjugation rate of GSH with CDNB higher than that found in the absence of reducing treatment, suggesting the presence of a significant, not spurious amount of oxidized e-GST (Figure 2a). Surprisingly, this additional activity was not recovered in isolated erythrocytes, but only in serum (Figure 2a). In a recent reinvestigation of the possible presence of GSTP1-1 in human serum, we concluded that it was absent or below the detection limit of the usual spectrophotometric assay,12 but we did not verify the possible occurrence of oxidized GSTP1-1. So far, no data are available in the literature concerning the presence of oxidized GSTP1-1 in bovine blood. Further experiments to clarify this point gave surprising results. In fact, the addition of NBDHEX, a strong and specific inhibitor of e-GST and other GST isoenzymes belonging to the alpha, pi and mu classes,13 did not suppress this additional GST activity (Figure 2b), and curiously the activity was also found in the absence of GSH (Figure 2a). Thus, the additional activity observed after reduction with DTT cannot be explained by the presence of oxidized GST in the samples.

View Article: PubMed Central - PubMed

ABSTRACT

Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans.

No MeSH data available.