Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B1 in human lung: differential cellular distribution and lack of significance of the GSTM1 genetic polymorphism

Richard K. Stewart1, Graeme B.J. Smith1, Patty J. Donnelly1, Ken R. Reid3, Dimitri Petsikas3, A.Alan Conlan3,4 and Thomas E. Massey1,2,5

1 Department of Pharmacology and Toxicology,
2 Department of Medicine and
3 Department of Surgery, Queen's University, Kingston, Ontario K7L 3N6, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies suggest that aflatoxin B1 (AFB1), a mycotoxin produced by certain Aspergillus species, may play a role in human respiratory cancers in occupationally-exposed individuals. AFB1 requires bioactivation to the corresponding exo-8,9-epoxide for carcinogenicity, and glutathione S-transferase (GST)-catalyzed conjugation of the epoxide with glutathione (GSH) is a critical determinant of susceptibility to AFB1. Of the purified human GST enzymes studied, the polymorphic hGSTM1-1 has the highest activity towards AFB1 exo-epoxide. The influence of the GSTM1 polymorphism on AFB1–GSH formation, as well as the abilities of cytosols from preparations enriched in different isolated lung cell types to conjugate AFB1-epoxides, were examined. In whole-lung cytosols from patients undergoing clinically indicated lobectomy, GSTM1 genotype correlated with GSTM1 phenotype as determined by [3H]trans-stilbene oxide conjugation: GSTM1-positive = 295 ± 31 pmol/mg/h (n = 6); GSTM1-negative = 92.8 ± 23.3 pmol/mg/h (n = 4) (P < 0.05). In contrast, conjugation of microsome-generated [3H]AFB1-epoxides with GSH was low and variable between patients, and did not correlate with GSTM1 genotype: GSTM1-positive = 11.9 ± 8.1, 111 ± 66 and 510 ± 248 fmol/mg/h (n = 6); GSTM1-negative = 15.3 ± 16.7, 167 ± 225 and 540 ± 618 fmol/mg/h (n = 4) (for 1, 10 and 100 µM [3H]AFB1, respectively). GSH conjugates of AFB1 exo-epoxide and the much less mutagenic stereoisomer AFB1 endo-epoxide were produced in a ratio of ~1:1 in cytosols from both whole lung and isolated cells. Total cytosolic AFB1-epoxide conjugation was significantly higher in fractions enriched in alveolar type II cells (3.07 ± 1.61 pmol/mg/h) than in unseparated lung cells (0.143 ± 0.055 pmol/mg/h) or fractions enriched in alveolar macrophages (0.904 ± 0.319 pmol/mg/h; n = 4) (P < 0.05). Furthermore, AFB1–GSH formation and percentage of alveolar type II cells in different cell fractions were correlated (r = 0.78, P < 0.05). These results demonstrate that human lung GSTs exhibit very low conjugation activity for both AFB1-8,9-epoxide stereoisomers, and that this activity is heterogeneously distributed among cell types, with alveolar type II cells exhibiting relatively high activity. Of the GSTs present in human peripheral lung which contribute to AFB1 exo- and endo-epoxide detoxification, hGSTM1-1 appears to play at most only a minor role.

Abbreviations: AFB1, aflatoxin B1; CDNB, 1-chloro-2,4-dinitrobenzene; EDTA, ethylenediaminetetraacetic acid; GSH, glutathione; GST, glutathione S-transferase; HEPES, N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; tSBO, trans-stilbene oxide.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aflatoxin B1 (AFB1) is a cytotoxic and carcinogenic mycotoxin produced by strains of the fungus Aspergillus flavus and related species. These fungi are ubiquitous, and under favourable conditions can grow on a wide variety of agricultural commodities. Though the liver is considered the primary target of AFB1 toxicity, the human lung can be exposed to AFB1 through inhalation of respirable contaminated grain dusts (1). In certain agriculture-related occupational settings, such exposure has been implicated in respiratory cancer (2) and liver cancer (3), and clinical studies have revealed AFB1–albumin adducts in the sera of animal feed processing workers (4). Additionally, AFB1 and/or metabolites have been found in lung tumour tissues of workers who died of lung carcinoma (5,6). The presence of DNA adducts suggests that the human lung also may be at risk of AFB1 toxicity after ingestion of contaminated foods (7,8).

For toxicity to occur, AFB1 requires bioactivation to exo and endo stereoisomers of AFB1-8,9-epoxide, although only AFB1 exo-epoxide binds appreciably to DNA (9). Glutathione S-transferases (GSTs) catalyze conjugation of AFB1-8,9-epoxides with reduced glutathione (GSH) to form AFB1 exo- and endo-epoxide–GSH conjugates (Figure 1Go). Thus, GSTs play a critical role in the protection of tissues from the deleterious effects of bioactivated AFB1 (10), and tissues vary considerably in both GST concentration and distribution of specific GST isoforms (11).



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Fig. 1. Bioactivation of AFB1 to exo- and endo-epoxides, and subsequent GST-catalyzed conjugation with GSH. PHS, prostoglandin H synthase; LOX, lipoxygenases.

 
In humans, the GST with the highest activity toward AFB1 exo-epoxide is the polymorphic hGSTM1-1 (12,13), which is absent in ~50% of individuals in most human populations (14). This suggests that AFB1-exposed individuals lacking the beneficial effects of hGSTM1-1 may be at elevated risk. Indeed, some reports suggest that the GSTM1 genetic polymorphism may affect AFB1 detoxification in human liver (15,16). In contrast to the liver, the lung is composed of many different cell types (17), and expression of GSTs in different human lung cell types is heterogeneous (1820). Thus, certain cell types with low levels of GSTs or lacking specific GST isoforms may be at higher risk of AFB1 toxicity. In the present study, the abilities of GSTs in peripheral human lung to detoxify bioactivated AFB1 were characterized, and the significance of the GSTM1 genetic polymorphism assessed. Additionally, cytosols prepared from freshly isolated human lung cells were used to investigate the abilities of different cell types to conjugate AFB1-8,9-epoxides.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
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 References
 
Chemicals
Chemicals were obtained as follows: generally labeled [3H]AFB1(16.9 Ci/mmol; >99.2% purity) from Moravek Biochemicals (Brea, CA); generally labeled [3H]trans-stilbene oxide (tSBO; 15 Ci/mmol; >99% purity) from American Radiolabeled Chemicals (St Louis, MO); glucose-6-phosphate, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP), 1-chloro-2,4-dinitrobenzene (CDNB), ethylenediaminetetraacetic acid (EDTA), deoxyribonuclease (DNase) type I, protease type XIV, RPMI-1640 medium and trypsin inhibitor type II-S from Sigma (St Louis, MO); Percoll from Pharmacia (Dorval, PQ); bovine serum albumin and N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (HEPES) from Boehringer Mannheim (Dorval, PQ); Taq polymerase, deoxynucleoside triphosphates, MgCl2 and polymerase chain reaction (PCR) buffer from Gibco BRL, Life Technologies (Gaithersburg, MD). Authentic AFB1–GSH, used as a high-performance liquid chromatography (HPLC) standard, was generously provided by Dr Gordon E.Neal and Dr David J.Judah (MRC Laboratories, Surrey, UK). AFB1 exo-epoxide–GSH standard, used for chiral HPLC analysis, was kindly supplied by Dr T.M.Harris (Vanderbilt University, Nashville, TN). All other chemicals were of reagent grade and were obtained from common commercial sources.

Animals and subcellular fractions
Male New Zealand white rabbits (2.0–2.5 kg), male Sprague–Dawley rats (250–300 g) and male CD-1 mice (25–30 g) were obtained from Charles River (LaPrairie, PQ). Animals were housed in individual cages on a 12 h light–dark cycle and fed Purina chow and water ad libitum. Animals were acclimatized to housing conditions for at least 1 week prior to experiments. Rats and mice were killed by decapitation, and rabbits received a single injection of sodium pentobarbital (78 mg/kg, 65 mg/ml) plus heparin (10 mg/kg, 50 mg/ml in saline) in a marginal ear vein. Liver microsomes and 100 000 g supernatants (cytosolic fractions) were prepared by differential centrifugation as described previously (21).

Human lung tissue procurement
Human lung tissue was obtained in accordance with procedures approved by the Queen's University Health Sciences Research Ethics Board. Following informed consent, sections of peripheral lung tissue (46 ± 18 g, mean ± SD, n = 10) devoid of macroscopically visible tumours, were removed during clinically indicated lobectomies. Immediately after removal, tissues were placed in 0.9% NaCl solution and stored on ice. Elapsed time between surgical removal of lung tissue and processing was ~20 min. Lung specimens were rinsed of blood with ice-cold HEPES-buffered salt solution, weighed and sections removed for fixation and hematoxylin and eosin staining (22) to confirm the absence of microscopic tumours. Sections from each tissue were also removed and frozen in liquid nitrogen for DNA isolation and PCR analysis. Patients were characterized with respect to age, sex, smoking history, possible occupational exposure to carcinogens, drug treatment 1 month prior to surgery and surgical diagnosis (Table IGo).


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Table I. Patient demographics and GSTM1 genotype
 
DNA isolation and GSTM1 genotyping
DNA was isolated from lung tissue by proteinase digestion followed by phenol–chloroform extraction and ethanol precipitation (23). PCR amplification of GSTM1 was performed as described previously (24) and PCR products from coamplification of GSTM1 and the ß-globin gene (positive control) were resolved on an ethidium-bromide-stained 3% agarose gel. Control DNA samples from GSTM1-positive and GSTM1-negative subjects were kindly provided by Dr D.A.Bell (National Institute of Environmental Health Sciences, Research Triangle Park, NC).

Preparation of human whole-lung cytosols
Whole-lung cytosols (100 000 g supernatants) were prepared by differential centrifugation as described previously (21), frozen in liquid nitrogen and stored at –70°C for up to 2 months. Following thawing, cytosols were concentrated using an Amicon model 8050 stirred ultrafiltration cell with a 10 000 Da MW cut-off membrane, at 30 p.s.i. N2 and 4°C. Cytosols were then dialyzed at 4°C for 22 h (12–14 kDa cut-off) against two changes of 1 l of 0.1 M potassium phosphate buffer (pH 7.4) containing 1.15% (w/v) KCl. Protein concentrations were determined by the method of Lowry et al. (25).

Human lung cell isolation and preparation of cell cytosols
Human lung cells were isolated by a previously published method (26,27). Briefly, following protease digestion, an aliquot of the unseparated cell suspension (cell digest) was saved, and the remaining cells underwent centrifugal elutriation using a Beckman J2-21M/E centrifuge with a JE-6B elutriation rotor. Three elutriation fractions were collected: the first (E1) contained primarily red blood cells and cellular debris; the second (E2) contained large numbers of alveolar type II cells, an aliquot of which underwent further enrichment; and the third (E3) contained a high proportion of alveolar macrophages.

Alveolar type II cells were further enriched from E2 by Percoll density gradient centrifugation. Cells were layered on a 38% (v/v) Percoll solution in RPMI-1640 medium and centrifuged at 1000 g for 15 min. Alveolar type II cells remained at the top of the Percoll solution, and the bottom layer consisted largely of neutrophils and red blood cells.

Viability and yield of cells were estimated by trypan blue exclusion on a hemocytometer (28). Using light microscopy, identification of alveolar type II cells was accomplished with the modified Papanicolaou method (29), which stains phospholipid-containing cytoplasmic lamellar bodies; alveolar macrophages were identified by their large size, round cell shape and nucleus, and the absence of stained cytoplasmic granules. Cell fractions were frozen in liquid nitrogen and stored at –70°C prior to subsequent preparation of cytosols. Cell fractions were thawed, lyzed by sonication, and cytosols prepared using differential centrifugation with Beckman Ultra-Clear micro centrifuge tubes (8x49 mm) as described previously (21,30).

[3H]tSBO assay to determine GSTM1 phenotype
[3H]tSBO conjugation activities were measured as described (31) with some modifications. Each 100 µl reaction mixture contained 90 µl dialyzed whole-lung cytosol diluted to 1–2 mg/ml with 0.1 M potassium phosphate buffer (pH 7.4), 5.0 µl GSH (final concentration, 5.0 mM) and 5 µl [3H]tSBO (final concentration 50 µM, 64 µCi/µmol). Incubations were carried out for 30 min at 37°C, followed by centrifugation at 200 g for 15 min, addition of 400 µl hexanol and further centrifugation (200 g, 15 min). Aliquots of aqueous phase containing [3H]tSBO–GSH were then removed for quantitation by liquid scintillation spectroscopy.

Measurement of CDNB conjugation activity in human whole-lung cytosols
GST-catalyzed conjugation of CDNB in human whole-lung cytosols was measured as described by Habig et al. (32), with some modifications. CDNB (60 µl of 50 mM in absolute ethanol), 1 mg/ml human lung cytosol (60 µl in 0.1 M potassium phosphate buffer pH 6.5) and 50 mM GSH (60 µl in 0.1 M potassium phosphate buffer pH 6.5) were mixed with 0.1 M potassium phosphate buffer (pH 6.5) plus 0.1 M EDTA in a total volume of 3.0 ml, and absorbance measured at 340 nm for 3 min.

Cytosolic incubations with [3H]AFB1
Incubations were carried out using glass tubes in the dark at 37°C in a shaking water bath. Each 1.0 ml reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.4) plus 1.15% (w/v) KCl, rabbit liver microsomes (2 mg protein), human whole-lung cytosol (16 ± 8 mg protein, mean ± SD) or human lung cell cytosol (4 ± 3 mg protein), 10 mM glucose-6-phosphate, 5.0 mM MgCl2, 1.0 mM EDTA, 5.0 mM GSH, 2 U glucose-6-phosphate dehydrogenase and 1.25 mM NADP. Incubations with whole-lung cytosol contained 1.0, 10 or 100µM [3H]AFB1 (7000, 700 or 70 µCi/µmol, respectively) dissolved in 10 µl dimethyl sulfoxide, while those with cytosol from isolated lung cells contained 10µM [3H]AFB1 (1500 µCi/µmol). Following 5 min of pre-incubation, reactions were initiated by addition of [3H]AFB1,and incubated for 1 h for whole-lung cytosols or 2 h for lung cell cytosols. Control incubations without human lung cytosolic protein were carried out to assess non-human GST catalysis (i.e. contribution of membrane-bound GST present in rabbit liver microsomes, or potential contamination of microsomes with rabbit liver cytosol), and spontaneous reaction of AFB1-epoxides with GSH.

Prior to determining the ability of human lung GSTs to conjugate the two AFB1-epoxide stereoisomers, it was necessary to confirm that the rabbit liver microsomal system used to generate the epoxides, produced similar amounts of AFB1 exo and endo stereoisomers. Incubations were conducted as described above, except 2 mg rabbit liver microsomal protein were combined with mouse, rat or rabbit liver cytosol (1 and 10 mg protein each) and 100 µM [3H]AFB1 (70 µCi/µmol). Mouse liver cytosolic GSTs possess high activity towards AFB1 exo-epoxide relative to AFB1 endo-epoxide (12), and though activity is considerably lower, rat liver cytosolic GSTs demonstrate stereospecificity for AFB1 endo-epoxide (12). We showed previously that rabbit liver cytosolic GSTs catalyze the GSH conjugation of both AFB1-epoxide isomers (33). Reactions were terminated by addition of 2.0 ml ice-cold chloroform, followed by mixing. Incubates were then centrifuged at 200 g for 15 min at 4°C, and the aqueous layers removed for storage at –20°C until analysis by HPLC.

HPLC analysis of [3H]AFB1–GSH
Total [3H]AFB1–GSH conjugate (i.e. not distinguishing between AFB1 exo-epoxide–GSH and AFB1 endo-epoxide–GSH) was isolated by reverse-phase HPLC based upon the method of Raj et al. (34). A 250 µl aliquot of aqueous phase was eluted isocratically with mobile phase consisting of 30% methanol in distilled water and 0.03% acetic acid, at a flow rate of 1.5 ml/min, utilizing a Waters µBondapak C18 column (3.9x300 mm). Peaks were detected by absorbance at 365 nm with a Waters Model 481 LC spectrophotometer, and 1.0 ml fractions were collected using a Gilson 203 fraction collector. Quantitation of [3H]AFB1–GSH conjugate was carried out by liquid scintillation spectoscopy, using a Beckman LS 3800 liquid scintillation counter and Fisher ScintiVerse BD scintillation cocktail. Identification of [3H]AFB1–GSH was established by co-elution of authentic AFB1–GSH standard.

AFB1 exo- and endo-epoxide–GSH conjugates were separated by a published method (35), and stereoisomer identity determined as described previously (33).

Statistical analysis
Data are presented as means ± SD. Student's t-test was used to compare tSBO and AFB1-epoxide conjugation activities from GSTM1-positive and GSTM1-negative patients and to assess smoking history effect on AFB1–GSH formation. Statistically significant differences were determined by repeated measures design one-way analysis of variance (ANOVA) for the isolated lung cell data, or two-way ANOVA with the whole-lung data, followed by the Tukey–Kramer multiple comparisons test. Pearson linear correlation was used to test the relationships between GST-catalyzed AFB1–GSH formation and percentage alveolar type II cells or percentage alveolar macrophages, and between CDNB conjugation and AFB1–GSH conjugation. A P-value less than 0.05 was considered statistically significant in all cases.


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Whole-lung cytosol experiments
Of the total of 14 patients, seven were GSTM1-positive and seven were GSTM1-negative (Table IGo). Conjugation of [3H]tSBO in whole-lung cytosols of GSTM1-positive individuals (295 ± 31 pmol/mg/min; n = 6) was ~3-fold higher than in cytosols from GSTM1-negative individuals (92.8 ± 23.3 pmol/mg/min; n = 4, P < 0.05) (Figure 2Go).



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Fig. 2. GSH-conjugation of [3H]tSBO in whole peripheral human lung cytosols from 10 patients. Mean values from triplicate analyses are shown (mean intra-assay variability < 16%).

 
Human whole-lung GSH conjugation of bioactivated [3H]AFB1 required the presence of cytosol and was AFB1 concentration dependent. Rates of conjugate formation were very low (Figure 3Go), with means of 13.3 ± 11.5 fmol/mg/h at 1 µM [3H]AFB1, 133 ± 142 fmol/mg/h at 10 µM [3H]AFB1 and 522 ± 402 fmol/mg/h at 100 µM [3H]AFB1. Conjugate formation in the GSTM1-positive (n = 6) and GSTM1-negative (n = 4) groups was not significantly different (P > 0.05): GSTM1-positive 11.9 ± 8.1, 111 ± 66 and 510 ± 248 fmol/mg/h; GSTM1-negative 15.3 ± 16.7, 167 ± 226 and 540 ± 618 fmol/mg/h (for 1.0, 10 and 100 µM [3H]AFB1, respectively).



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Fig. 3. Formation of [3H]AFB1–GSH in cytosolic fractions prepared from whole peripheral human lung from 10 patients. Mean values from duplicate HPLC analyses are shown (<5% variability between analyses).

 
[3H]AFB1-epoxide conjugation also was not significantly different between current smokers (n = 6) and non-smokers or former smokers (n = 4, P > 0.05): current smokers 15.7 ± 14.1, 165 ± 177 and 534 ± 480; non-smokers or former smokers 9.58 ± 5.92, 84.3 ± 58.0 and 503 ± 315 (for 1.0, 10 and 100 µM [3H]AFB1, respectively).

Rabbit liver microsomes generated AFB1 exo- and endo-epoxides in a ratio of ~1:1. Chiral HPLC analysis of whole human lung cytosol incubations with 100 µM [3H]AFB1 bioactivated by rabbit liver microsomes showed that AFB1 exo-epoxide–GSH and AFB1 endo-epoxide–GSH were produced in a ratio of ~1:1 (data not shown).

CDNB conjugation activity was 0.49 ± 0.20 µmol/mg/min in human whole-lung cytosols, and activities for the different patients did not significantly correlate with [3H]AFB1–GSH formation (P > 0.05, Pearson linear correlation analysis, r = 0.31, 0.43 and 0.56 for correlation between CDNB conjugation and [3H]AFB1–GSH production with 1.0, 10 and 100 µM [3H]AFB1, respectively; n = 10).

Isolated lung cell cytosol experiments
The E2-Percoll top fraction, enriched in alveolar type II cells (64.0 ± 5.8%, Table IIGo), also contained macrophages and polymorphonuclear leukocytes, each comprising <10% of the cells. The third elutriation fraction (E3), enriched in alveolar macrophages (54.2 ± 18.1%), also consisted of ~10–25% type II cells and few polymorphonuclear leukocytes. Cell viability in all fractions was at least 88% as determined by trypan blue exclusion.


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Table II. Summary of lung cell isolations from which cytosolic fractions were prepareda
 
The highest [3H]AFB1–GSH conjugation activity was in cytosols from the alveolar type II cell enriched fraction (3.07 ± 1.61 pmol/mg/h) (Figure 4Go) and was significantly different from all other isolated cell fractions (P < 0.05). Furthermore, a significant correlation was observed between AFB1–GSH formation and percentage of alveolar type II cells in different cell fractions (r = 0.78, P < 0.05), with no significant correlation being found with percentage of alveolar macrophages r = –0.16, P > 0.05) (Figure 5Go). As in the case of whole lung, isolated cell cytosols produced [3H]AFB1 exo- and endo-epoxide–GSH conjugate stereoisomers in a ratio of ~1:1.



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Fig. 4. Formation of [3H]AFB1–GSH in cytosols from isolated human lung cells. E1 (elutriation fraction 1) contained primarily red blood cells and cellular debris; E2-Percoll top consisted mostly of alveolar type II cells; E2-Percoll bottom contained primarily neutrophils and some red blood cells; and E3 contained mostly alveolar macrophages. Patients 11, 12, 13 and 14 were GSTM1-negative, -positive, -negative and -negative, respectively. Mean values from duplicate HPLC analyses are shown (<5% variability between analyses).

 


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Fig. 5. Pearson linear correlation analysis between [3H]AFB1–GSH formation in cytosols from isolated human lung cells and (A) percent alveolar type II cells in each fraction; (B) percent alveolar macrophages in each fraction. Some data points are from cells in partially enriched fractions not shown in Figure 4Go.

 
Although the number of tissue samples used for cell isolation was small, the lack of association between [3H]AFB1–GSH formation and GSTM1 genotype in whole-lung cytosols appeared to be supported by the isolated cell results in that no clear difference was apparent between the one GSTM1-positive (no. 12) and three GSTM1-negative patients (nos 11, 13 and 14) (Figure 4Go).


    Discussion
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 Abstract
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 Materials and methods
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 References
 
Epidemiological and clinical studies suggest that the human respiratory system can be at risk of AFB1 toxicity following both inhalation (2,46) and ingestion (7,8). We have shown previously that lipoxygenases and prostaglandin H-synthase activate AFB1 via co-oxidation in human lung, with cytochrome P450-catalyzed oxidation a quantitatively much less important bioactivation pathway (27). Other investigators have also found evidence showing very low levels of human lung microsomal bioactivation of AFB1 (36). Since GST-catalyzed conjugation of AFB1 exo- and endo-epoxides with GSH is a critical detoxification pathway, the characterization and localization of human lung GSTs with respect to these electrophilic toxicants were the aims of the present work. Overall, human lung cytosol had very low AFB1-epoxide conjugating activity, with rabbit lung activity being ~350-fold higher (33).

Determining the ratio of AFB1-epoxides that are detoxified in a given tissue or cell has important toxicological implications, since only the exo-epoxide interacts significantly with DNA (9). Though not highly genotoxic, the endo-epoxide could conceivably contribute to acute toxicity by binding to critical cellular proteins. We showed previously that rabbit lung GSTs can detoxify AFB1 exo-epoxide, but not AFB1 endo-epoxide (33). Thus, human lung differs from rabbit lung, since the former detoxified both AFB1-epoxides to their corresponding GSH conjugates to similar degrees, in both whole lung and isolated cell cytosols.

The cytosolic GST enzymes known to be present in human lung belong to three main classes, {alpha}, µ and {pi}, which represent 30, 10 and 60% of total GST protein, and 3, 3 and 94% of total CDNB conjugation activity, respectively (37). The {pi}-class GST, designated hGSTP1-1, exhibits negligible activity towards AFB1-epoxides (12,13). In the present study, the lack of correlation between CDNB conjugating activity and AFB1–GSH formation confirms the minimal contribution of hGSTP1-1 towards AFB1-epoxide detoxification in the human lung. The {alpha}-class subunits hGSTA1 and A2 have been detected in human lung (38), and Singhal et al. (37) purified three {alpha}-class GST proteins from human lung tissue. Immunostaining of the {alpha}-class GSTs showed wide variability between individuals, both in intensity and distribution, with localization occurring in bronchial, bronchiolar and alveolar epithelium and in alveolar macrophages (18). Purified hGSTA1-1 and hGSTA2-2 have measurable but low activity with AFB1 exo- and endo-epoxides as substrates (12), although experiments using recombinant hGSTA1-1 and hGSTA2-2 showed negligible activity toward AFB1 exo-epoxide (13). Thus, {alpha}-class GSTs may have been contributors to the low levels of AFB1-epoxide conjugation we observed.

In the case of the µ-class GSTs in human lung, all five known subunits have been detected: hGSTM1, M2, M3, M4 and M5 (3840). Immunohistochemical localization of three µ-class GSTs in human lung showed very low or absent GSTM1 in bronchial and bronchiolar epithelium, low levels of GSTM2 in the terminal bronchiolar epithelium and alveolar type II cells, and GSTM3 which varied between individuals from minimal to intense in bronchial and bronchiolar epithelium, bronchial glands and in bronchial and vascular smooth muscle (18). The GSTM1 gene has attracted particular interest with respect to defense against chemical carcinogens. It is polymorphic in humans, with individuals possessing genetically-determined combinations of the GSTM10, M1a and M1b alleles [the 1a and 1b proteins differ only by one amino acid, and demonstrate similar catalytic properties (41)]. The deficiency in enzyme activity is caused by inherited homozygous deletion of the GSTM1 gene, which occurs with a frequency of ~50% in most populations (42).

Several studies with human liver have investigated the potential relationship between AFB1 carcinogenicity and the GSTM1 polymorphism. Liver preparations from patients with high hGSTM1-1 activity inhibited the formation of AFB1–DNA adducts more than those from individuals with low hGSTM1-1 activity (15), and purified hGSTM1a-1a had the highest activity for AFB1-epoxides, relative to other human GSTs (hGSTA1-1, A2-2, M3-3 and P1-1) (12). Furthermore, studies with recombinant human GSTs showed hGSTM1b-1b to catalyze the conjugation of AFB1 exo-epoxide with greatest efficiency (13). These results are consistent with the reported detection of AFB1–GSH in cultured primary hepatocytes from GSTM1-positive patients only (16), but contradict the findings of Slone et al. (43), who failed to find a correlation between immunochemically and enzymatically detected GSTµ expression and AFB1-epoxide conjugation in human liver cytosols. Our results show that hGSTM1-1 does not contribute significantly to the detoxification of AFB1-epoxides in human peripheral lung.

The most abundant µ-class GST throughout the conductive and peripheral human lung, hGSTM3-3, exhibits low activity for AFB1 exo- and endo-epoxides (12). Interestingly, a polymorphism in hGSTM3-3 has been discovered, but the activities of the different phenotypes (hGSTM3a-3a, M3b-3b and M3a-3b) have yet to be characterized (44). Furthermore, data suggest that expression of GSTM3 is influenced by GSTM1 genotype, with GSTM1-negative individuals expressing significantly less GSTM3-3 than the other GSTM1 genotypes (45). If GSTM3-3 expression and GSTM1 genotype are linked, and if GSTM3-3 is important in AFB1 detoxification in human lung, then a correlation between GSTM1 genotype and AFB1–GSH formation in the present study would have been expected. However, this was not the case. The other known µ-class GSTs in human lung, hGSTM2-2, M4-4 and M5-5, may be important in detoxifying electrophilic carcinogens, but activities towards such substrates (including AFB1-epoxides) are currently unknown.

Our observed lack of correlation between AFB1-epoxide conjugation and smoking history is consistent with the previously reported lack of difference in immunoreactivity for several GSTs (45) and CDNB conjugating activity (46) in human lung tissue from smokers versus non-smokers.

Cytosolic GSTs in alveolar type II cells had higher activity for conjugating bioactivated AFB1 than did other cell fractions. Some studies have shown the presence of {alpha}-class and {pi}-class GSTs in type II cells (18), but these enzymes display low or undetectable conjugating activity with AFB1-epoxides (12,13), suggesting that the activity found in this cell type is due to the concentration of GST isoforms with relatively low activity for AFB1 epoxides. Alveolar type II cells represent ~16% of the cells of human lung (47), and demonstrate relatively high biotransformation activities compared with other human lung cell types (18,26,48,49). Although CYP3A4 can bioactivate AFB1 and is present in type II cells, we demonstrated previously that activation of AFB1 to AFB1 exo-epoxide occurs predominantly via prostaglandin H synthase and lipoxygenase co-oxidation in both alveolar type II cells and alveolar macrophages (27). The present study showed that AFB1 exo- and endo-epoxide conjugating activity occurs at low levels in alveolar macrophages, which possess the {alpha}-class GSTs, hGSTP1-1, hGSTM3-3 and hGSTM1-1 (18,20). Thus, cytosolic GSTs affect the activation/detoxification balance such that detoxification is favoured more in type II cells than in macrophages. This is consistent with our earlier finding that, following in vitro exposure of intact human lung cells to AFB1, levels of AFB1–DNA binding were significantly higher in macrophages than in type II cells.

The {theta}-class hGSTT1-1, which is also polymorphic (50), has AFB1 exo-epoxide conjugating activity which is less than that of hGSTM1-1, but greater than that of hGSTP1-1 and the {alpha}-class GSTs (13). Low levels of hGSTT1-1 mRNA have been found in some human lung samples (51), but it is expressed at higher levels in red blood cells. Therefore, if hGSTT1-1 contributes appreciably to AFB1 detoxification in the lung, AFB1–GSH formation should have been high in the cell fraction with the highest concentration of erythrocytes, elutriation fraction E1. However, this was not the case.

In conclusion, human lung possesses very low conjugation activity for both AFB1-8,9-epoxide stereoisomers, and this activity is heterogeneously distributed among cell types. Contributions of several GSTs, including those of the {alpha}-class and µ-class other than hGSTM1-1, probably account for the conjugation activity present. The human lung may be at risk of AFB1 toxicity due to its limited ability to conjugate AFB1-epoxides with GSH.


    Acknowledgments
 
The authors wish to thank Dr Douglas A.Bell for his assistance with establishing the GSTM1 genotyping protocol and for helpful discussions regarding this study. The authors would also like to thank Ms Sophia L.Ali for assisting with tissue preparation, and Ms Barbara J.Veley for coordinating the acquisition of lung tissues and organizing patient histories. This work was supported by Medical Research Council of Canada (MRC) grant MT-10382.


    Notes
 
4 Present address: Department of Surgery, University of Massachusetts, Worchester, MA, USA Back

5 To whom correspondence should be addressed Email: masseyt{at}post.queensu.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 13, 1999; revised May 6, 1999; accepted May 17, 1999.