Preferential Glutathione Conjugation of a Reverse Diol Epoxide Compared with a Bay Region Diol Epoxide of Benzo[a]pyrene in Human Hepatocytes (original) (raw)
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Chemico-biological interactions, 2017
Polycyclic aromatic hydrocarbons (PAH) are environmental carcinogens implicated as causes of cancer in certain industrial settings and in cigarette smokers. PAH require metabolic activation to exert their carcinogenic effects. One widely accepted pathway of metabolic activation proceeds through formation of "bay region" diol epoxides which are highly reactive with DNA and can cause mutations. Phenanthrene (Phe) is the simplest PAH with a bay region and an excellent model for the study of PAH metabolism. In previous studies in which [D10]Phe was administered to smokers, we observed higher levels of [D10]Phe-tetraols derived from [D10]Phe-diol epoxides in subjects who were null for the glutathione-S-transferase M1 (GSTM1) gene. We hypothesized that Phe-epoxides, the primary metabolites of Phe, were detoxified by glutathione conjugate formation, which would result ultimately in the excretion of the corresponding mercapturic acids in urine. We synthesized the four stereoisomer...
Archives of Biochemistry and Biophysics, 1999
Benzo[a]pyrene-(7R,8S)-diol (9S,10R)-epoxide [(؉)anti-BPDE] is believed to be the activated form of the widely spread environmental pollutant benzo-[a]pyrene. Glutathione (GSH) S-transferase (GST)catalyzed conjugation of (؉)-anti-BPDE with GSH is an important mechanism in its cellular detoxification. Here, we report that the GSH conjugate of (؉)anti-BPDE [(؊)-anti-BPD-SG] is a potent inhibitor (K i 15 M) of class Mu human GST isoenzyme, which, among human liver GSTs, is a highly efficient detoxifier of (؉)-anti-BPDE. Thus, the inhibition of GST activity by (؊)-anti-BPD-SG may hinder GSH conjugation of (؉)-anti-BPDE, unless the conjugate is metabolized and/or eliminated. The results of the present study show that ␥-glutamyltranspeptidase (␥-GT) can metabolize (؊)-anti-BPD-SG at a rate of about 0.29 nmol/min/mg protein. Our studies also show that (؊)-anti-BPD-SG is transported across the human canalicular liver plasma membrane (cLPM) in an ATP-dependent manner at a rate of about 0.33 nmol/min/mg protein. The ATP-dependent transport of (؊)-anti-[ 3 H]BPD-SG across human cLPM follows Michaelis-Menten kinetics (K m 84 M; V max 0.33 nmol/ min/mg). In conclusion, the results of the present study suggest that both ␥-GT-mediated metabolism and ATP-dependent canalicular transport may be important steps in overall detoxification of (؉)-anti-BPDE in the human liver.
FEBS Letters, 1998
Epidemiological studies suggest that individuals differing in the expression of allelic variants of the human glutathione transferase (GST) Pi gene differ in susceptibility to chemical carcinogens such as polycyclic aromatic hydrocarbons (PAH). This study reports the catalytic efficiencies (k t /K m ) of two naturally occurring variants, GSTP1-1/I-105 and GSTP1-1/ V-105, towards a series of fjord-region diol epoxides representing potent biologically active PAH metabolites, and two GSTP1-1 mutants with Ala IHS and Trp IHS in the active site. The results indicate that individuals who are homozygous for the allele encoding GSTP1-1/V-105 might be more susceptible to PAH carcinogenesis due to other reasons than a reduced capacity for detoxifying diol epoxides.
Mutagenesis, 2008
The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the a-class selective 7chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.
Carcinogenesis, 1998
Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (k cat /K m ) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti-and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest k cat /K m value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of k cat /K m . With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased k cat /K m values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionyl-*Abbreviations: PAH, polycyclic aromatic hydrocarbons; CDNB, 1-chloro 2,4-dinitrobenzene; syn-and anti- CDE,4epoxide;h]anthracene-1,2-diol 3,4-epoxide.
Ecotoxicology and Environmental Safety, 2021
Polycyclic Aromatic Hydrocarbons (PAH) are a class of organic pollutants normally found as mixtures with effects often hard to predict, which poses a major challenge for risk assessment. In this study, we address the effects of Phenanthrene (Phe), benzo[b]fluoranthene (B[b]F) and their mixtures (2 Phe:1 B[b]F; 1 Phe: 1 B[b]F; 1 Phe: 2 B[b]F) over glutathione (GSH) synthesis and function in HepG2 cells. We analyzed the effects on cellular viability, ROS production, glutathione (GSH) levels, proteinS -glutathionylation (PSSG), the activity of glutathione peroxidase (GPx), glutathione-S-transferases (GST) and glutathione reductase (GR). Transcript (mRNA) levels of glutathione synthesis enzymes-glutathione cysteine ligase catalytical (GCLC) and modifying (GCLM) sub-units and glutathione synthetase (GS)-and Nrf2 translocation to the nucleus were analyzed. Phe showed a higher cytotoxicity (IC 50 = 130 µM after 24 h) than B[b]F related to a higher ROS production (up-to 50% for Phe). In agreement, GSH levels were significantly increased (up-to 3-fold) by B[b]F and were accompanied by an increase in the levels of PSSG, which is a mechanism that protect proteins from oxidative damage. The upregulation of GSH was the consequence of Nrf2 signaling activation and increased levels of GCLC, GCLM and GS mRNA observed after exposure to B[b]F, but not during exposure to Phe. Most interestingly, all mixtures showed higher cytotoxicity than individual compounds, but intriguingly it was the 1 Phe: 1B[b]F mixture showing the highest cytotoxicity and ROS production. GSH levels were not significantly upregulated not even in the mixture enriched in B[b]F. These results point to the role of GSH as a central modulator of PAH toxicity and demonstrate the idiosyncratic behavior of PAH mixtures even when considering only two compounds in varying ratios.
International Journal of Cancer, 1997
There is a growing need for short-term and cost-effective bioassay to assess the efficacy of potential chemo-preventive agents. We report that the induction of glutathione (GSH) S-transferase (mGSTP1-1) by a chemo-preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1-1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (؉)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(؉)-anti-BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mG-STP1-1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP-induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (؉)-anti-BPDE was far less than the contribution of mGSTP1-1 alone. Likewise, in the forestomach, the contribution of mGSTP1-1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BPinduced forestomach neoplasia revealed a good correlation between their chemo-preventive efficacy and their ability to induce mGSTP1-1 expression in the liver (r ؍ ؊0.89; p F 0.05) as well as in the forestomach (r ؍ ؊0.97; p F 0.05). Our results suggest that the induction of mGSTP1-1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP-induced cancer in a murine model. Int.
Aromatic Hydrocarbons; Comparison with Rat Liver and Lung
2008
The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electro-philic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]-fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]an-thracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When
Chemical Research in Toxicology, 2002
In this study, human glutathione transferases (GSTs) of alpha class have been assayed with the ultimate carcinogenic (-)-anti-and (+)-syn-diol epoxides (DEs) derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-diol epoxide of the planar benzo[a]pyrene [(+)anti-BPDE] in the presence of glutathione (GSH). In all DEs, the benzylic oxirane carbon reacting with GSH, possess R-absolute configuration. GSTA1-1 demonstrated activity with all DEs tested whereas A2-2 and A3-3 only were active with the DBPDE enantiomers. With GSTA4-4, no detectable activity was observed. GSTA1-1 was found to be the most efficient enzyme and demonstrated a catalytic efficiency (k cat /K m ) of 464 mM -1 s -1 with (+)-syn-DBPDE. This activity was about 7-fold higher than that observed with (-)-anti-DBPDE and more than 65-fold higher than previously observed with less complex fjord-region DEs. GSTA3-3 also demonstrated high k cat /K m with the DEs of DBP and a high preference for the (+)-syn-DBPDE enantiomer [190 vs 16.2 mM -1 s -1 for (-)-anti-DBPDE]. Lowest k cat /K m value of the active enzymes was observed with GSTA2-2. In this case, 30.4 mM -1 s -1 was estimated for (+)-syn-DBPDE and 3.4 mM -1 s -1 with (-)-anti-DBPDE. Comparing the activity of the alpha class GSTs with (-)-anti-DBPDE and (+)-anti-BPDE revealed that GSTA1-1 was considerable more active with the former substrate (about 25-fold). Molecular modeling studies showed that the H-site of GSTA1-1 is deeper and wider than that of GSTA4-4. This is mainly due to the changes of Ser212fTyr212 and Ala216fVal216, which cause a shallower active site, which cannot accommodate large substrates such as DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to (-)-anti-DBPDE is explained by the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The presence of GSTA1-1 in significant amounts in human lung, a primary target tissue for PAH carcinogenesis, may be an important factor for the protection against the harmful action of this type of potent carcinogenic intermediates. † Part of this study was presented at the 18th International
Biochemical and Biophysical Research Communications, 1997
Glutathione S-transferase (GST) Pi is a member of Allelic forms of hGSTP1-1 which differ from each one of the four major classes of mammalian cytosolic other by their catalytic properties and, structurally, GSTs (1-3). The enzyme, termed hGSTP1-1 in humans, by the amino acid(s) in position(s) 104 or (and) 113 has been extensively studied because of its overexare known to exist in human populations. The four pression in pre-neoplastic and tumor tissues. This bepossible isoforms of hGSTP1-1 with isoleucine or vahavior makes hGSTP1-1 useful as an early tumor line in position 104 and with alanine or valine in posimarker, but is also responsible for drug resistance of tion 113 were produced by site-directed mutagenesis many cancers (3-5). Of no less importance is the role of the cDNA followed by bacterial expression and puriof hGSTP1-1 in the metabolism and detoxification of fication of the proteins. Glutathione-conjugating activmany carcinogenic xenobiotics, among them the diol ity was measured with the diol epoxides of benzo(a)epoxides of polycyclic aromatic hydrocarbons (PAHs) pyrene and chrysene, as well as with the model subsuch as benzo(a)pyrene (6,7) or chrysene (8). Because of strate 1-chloro-2,4-dinitrobenzene. Isoenzymes with this activity, hGSTP1-1 is expected to afford protection