Activation and Inactivation of Carcinogenic ... - ACS Publications

Chem. Res. Toxicol. 1996, 9, 333-340

333

Activation and Inactivation of Carcinogenic Dihaloalkanes and Other Compounds by Glutathione S-Transferase 5-5 in Salmonella typhimurium Tester Strain NM5004 Tsutomu Shimada,*,† Hiroshi Yamazaki,† Yoshimitsu Oda,† Akira Hiratsuka,‡ Tadashi Watabe,‡ and F. Peter Guengerich§ Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku, Osaka 537, Japan, Department of Drug Metabolism and Molecular Toxicology, Tokyo College of Pharmacy, Tokyo 192-03, Japan, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennesee 37232 Received July 10, 1995X

A newly developed tester Salmonella typhimurium NM5004 strain was constructed by introducing a plasmid containing both rat GSH S-transferase (GST) 5-5 cDNA and the umuC′′lacZ operon into the host strain Salmonella typhimurium TA1535 and used to examine whether or not GST modified the genotoxic activities of several dihaloalkanes and other compounds. Twenty-nine chemicals that were suggested to be conjugated by GST were compared with regard to their abilities to induce umu gene expression and cause cytotoxicity responses in both the NM5004 strain and the original tester strain (S. typhimurium TA1535/ pSK1002, which is devoid of GST activity toward 1,2-epoxy-3-(4′-nitrophenoxy)propane). Ten chemicalss1,2-dibromoethane, N-(2,3-epoxypropyl)phthalimide, 1,3-dichloroacetone, CH2I2, 1,2epoxy-3-phenoxypropane, 2,3-epoxypropyl p-methoxyphenyl ether, 1-bromo-2-chloroethane, 1-bromo-2,3-dichloropropane, CH2BrCl, and CH2Br2swere found to enhance induction of umu gene expression in the NM5004 strain as compared with the TA1535/pSK1002 strain. 1,2Epoxy-3-(4′-nitrophenoxy)propane and 2,3-dibromo-1-chloropropane were inactivated by GST 5-5 in the NM5004 tester strain, although these chemicals were cytotoxic in both tester strains. Roles of GST 5-5 were also examined for the inactivation of reactive metabolites of several procarcinogens that were formed through oxidation by liver microsomes of polychlorinated biphenyl-treated rats. The results suggest that reactive metabolites (possibly epoxides) of aflatoxin B1, sterigmatocystin, 1,2-dihydro-1,2-dihydroxy-6-aminochrysene, and (+)- and (-)enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene could be trapped as inactivated GSH conjugates in the NM5004 strain. High-performance liquid chromatographic analysis suggested that exo-aflatoxin B1-8,9-oxide-GSH conjugate was formed during the oxidation of aflatoxin B1 by rat and human liver microsomes in the presence of GSH and several GST enzymes including purified rat theta class GST Yrs-Yrs and rat liver GST (a mixture of alpha and mu class enzymes). Thus, the present results support the view that the theta class rat GST 5-5 enzyme participates in the activation and inactivation of potential environmental carcinogenic chemicals. This newly developed NM5004 tester strain is of use in the elucidation of roles of GST 5-5 in transformations.

Introduction Multiple forms of glutathione S-transferase (GST)1 are found in subcellular fractions of mammals, and recent studies have established that soluble GST enzymes can be classified into four groups (alpha, mu, pi, and theta) on the basis of structural similarity of isolated genes (15). In general, GST enzymes act to inactivate toxic * Address correspondence to this author at the Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan. FAX: 81-6-972-2393. † Osaka Prefectural Institute of Public Health. ‡ Tokyo College of Pharmacy. § Vanderbilt University School of Medicine. X Abstract published in Advance ACS Abstracts, December 15, 1995. 1 Abbreviations: PCB, polychlorinated biphenyl mixtures containing 55% chlorine (Kanechlor 500); 6-AC, 6-aminochrysene; 6-AC-diol, trans1,2-dihydro-1,2-dihydroxy-6-aminochrysene; ENPP, 1,2-epoxy-3-(4′nitrophenoxy)propane; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQ, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline; MeIQx, 2-amino3,8-dimethylimidazo[4,5-f]quinoxaline; Trp-P-1, 3-amino-1,4-dimethyl5H-pyrido[4,3-b]indole; B[a]P-7,8-diol, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; AF, aflatoxin; GST, glutathione S-transferase.

0893-228x/96/2709-0333$12.00/0

chemicals, carcinogens, and mutagens by conjugating them with GSH; however, in several cases the enzymes participate in the formation of reactive GSH conjugates that alkylate cellular macromolecules (6, 7). For example, theta class rat GST 5-5 and alpha class human GST A1-1 have been shown to activate several dihaloalkanes to mutagenic metabolites in Salmonella typhimurium TA1535 and TA100, respectively, in which respective GST cDNA clones had been introduced (8-11). Recently, human theta class GSTT1 has been purified from liver, and the cDNA clone has been isolated and characterized (4, 12). Efforts have been made to determine if there are genetic polymorphisms in GSTT1 gene expression in humans, and extensive studies have indicated that about 40% of human populations are defective in expression of this GST gene (12, 13). The substrate specificities of human GSTT1 and rat GST 5-5 have been reported to be similar in terms of mutagenic activation © 1996 American Chemical Society

334 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

of several dihaloalkanes in S. typhimurium TA1535 tester strains in which the respective cDNA clones were introduced (14). Thus, roles of GST enzymes in the activation and inactivation of carcinogenic chemicals must be determined in order to understand the basis for mechanisms of susceptibilities of individual humans toward environmental carcinogens. This study was undertaken to further examine the roles of rat GST 5-5 in the activation and inactivation of environmental carcinogens using a new S. typhimurium tester strain (NM5004), which was constructed by introducing both rat GST 5-5 cDNA and the umuC′′lacZ operon into the host strain S. typhimurium TA1535. A total of 29 chemicals suggested to be conjugated with GSH by GST (7, 8, 11, 15-20) were selected and compared with regard to inducing umu gene expression and cytotoxicity responses in both NM5004 strain and the original tester strain, S. typhimurium TA1535/ pSK1002. We also report the roles of GST 5-5 in the inactivation of genotoxic metabolites of several procarcinogens including carcinogenic arylamines, mycotoxins, and polycyclic aromatic hydrocarbons. HPLC analysis in the formation of exo-aflatoxin (AF) B1-8,9-oxide-GSH conjugate after metabolism of AFB1 by rat and human liver microsomes in the presence of GSH and several GST enzymes is presented.

Experimental Procedures Chemicals. (()-1,2,3,4-Diepoxybutane, 1,2-epoxy-3-chloropropane, 1,4-dibromobutane, and AFB1 were purchased from Aldrich Chemical Co. (Milwaukee, WI). 1,2-Epoxy-3-(4′-nitrophenoxy)propane (ENPP) was from Wako Pure Chemical Co. (Osaka). AFM1 and AFQ1 were from Sigma Chemical Co. (St. Louis, MO). The exo-AFB1-8,9-epoxide-GSH conjugate was prepared as described (21). Other chemicals used were the same as described (11) or were purchased from Aldrich Chemical Co. Enzymes. Male Sprague-Dawley rats (weighing about 200 g) were obtained from Nihon Clea Co., Osaka. Rats were treated ip with polychlorinated biphenyls (PCB) (80 mg/kg, daily for 3 days) and were starved overnight before being killed. Liver microsomes were prepared and suspended in 10 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM EDTA and 20% glycerol (v/v) as described previously (22). Liver microsomes from human sample HL-4 (which is high in P450 3A4 content) were also prepared (23, 24). Rat liver GST (a mixture of alpha and mu class of enzymes) was purchased from Sigma. Rat liver theta class GST Yrs-Yrs, which is active in detoxicating reactive sulfate esters of carcinogenic polycyclic arylmethanols, was purified as described previously (25). Bacterial Tester Strains. S. typhimurium NM 5004 tester strain was constructed by the method described previously (26, 27).2 Briefly, the plasmid pSK1002 carrying the umuC′′lacZ gene operon was partially digested with the restriction enzymes SalI and EcoRI, and the resulting 11.3-kb EcoRI-SalI fragment containing the umuC′′lacZ operon was cloned into the EcoRISalI stuffer region of the GST 5-5 vector (pKK233-2) (8, 28). Selection and transformation of the chimeric plasmid (carrying both the umuC′′lacZ fusion gene and GST 5-5 gene) into S. typhimurium TA1535 were carried out as described previously.2 The tester strain S. typhimurium NM5004 thus obtained was shown to express GST 5-5 protein on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and had high GST activity when ENPP was used as a substrate.2 The original umu tester strain S. typhimurium TA1535/pSK1002 was constructed by the method described previously (26, 27). 2 Y. Oda, H. Yamazaki, R. Thier, B. Ketterer, F. P. Guengerich, and T. Shimada (1995), submitted for publication.

Shimada et al. Assay Methods. The genotoxicities of chemical carcinogens and mutagens were determined by measuring induction of an umu gene expression in the two Salmonella tester strains as described previously (26, 27, 29). Briefly, overnight cell cultures of tester strains were grown in TGA medium containing 20 µg of ampicillin/mL until the optical density of the bacteria at 600 nm was ∼0.3. The resulting bacterial suspension was incubated with chemicals (dissolved in Me2SO) at 37 °C for 2 h, and the expressed β-galactosidase activity was determined by the method of Miller (30) and expressed in those units or absorbance at 420 nm using o-nitrophenyl-β-D-galactopyranoside as a substrate. Cell toxicity was determined in the reaction mixture by measuring the optical density change at 600 nm. P450-dependent activation of procarcinogens to reactive metabolites that cause induction of an umu gene expression in tester strains S. typhimurium TA1535/pSK1002 and NM5004 was done with liver microsomes of PCB (Kanechlor 500, a polychlorinated biphenyl mixture containing 55% chlorine)treated rats as described previously (27, 29). Incubation mixtures consisted of rat liver microsomes (0.01 µM P450) with 1-5 µM procarcinogens in a final volume of 1.0 mL of 100 mM potassium phosphate buffer (pH 7.4) containing an NADPHgenerating system and 0.75 mL of bacterial suspension as described previously (29, 31). Incubations were carried out at 37 °C for 2 h and terminated by cooling the mixtures on ice. The expression of umu gene expression was monitored by measuring β-galactosidase activities as described above and expressed as units min-1 (mg of microsomal protein)-1. Data presented in this study were the means of duplicate or triplicate determinations, and the standard deviations in these values were less than 15% of the means. Detection of GSH Adducts and Other Metabolites of AFB1 in Systems Containing Purified Rat GST Enzymes by HPLC. AFB1 (20 µM) was incubated with liver microsomes (0.01 mg of protein) of PCB-treated rats or with human liver (sample HL-4) microsomes (0.1 mg of protein) described above in the presence of purified rat liver GST enzyme (0.05 mg of protein) and rat theta class GST Yrs-Yrs enzyme (0.5 µg of protein), 30 mM MgCl2, and 3.0 mM GSH. The mixtures (final volume of 0.25 mL) were incubated at 37 °C for 20 min. The reaction was terminated by adding 0.1 mL of 0.4 M HCO2H, and the mixtures were kept at -20 °C overnight. The formation of GSH adducts was determined by HPLC using endo- and exoAFB1-epoxide-GSH conjugates as standards. Separation was conducted with a column of Alltech Econosphere C18 (4.6 × 250 mm, Alltech, Deerfield, IL) and elution with a mixture of 43% (v/v) CH3OH in 0.1 M sodium phosphate buffer (pH 3.0) at a flow rate of 0.8 mL/min (32). Other Assays. P450 was estimated spectrally by the method of Omura and Sato (33). Protein contents were estimated by the method of Lowry et al. (34).

Results Comparison of Genotoxicity Activities of 29 Chemicals in S. typhimurium TA1535/pSK1002 and NM5004 Strains. Induction of umu gene expression and cytotoxicity by 10 chemicals was compared with the two tester strains in a dose-dependent manner (Figures 1 and 2). 1,2-Dibromoethane was found to be highly genotoxic and cytotoxic only in the NM5004 strain. Because this chemical showed severe cytotoxic activity, the umu gene expression was decreased when higher concentrations of 1,2-dibromoethane were used (Figure 1). A similar pattern was also noted with 1-bromo-2-chloroethane (Figure 2). 1,3-Dichloroacetone, CH2I2, 1-bromo-2,3dichloropropane, CH2BrCl, and CH2Br2 were not cytotoxic in either tester strain but were highly genotoxic in only the NM5004 strain (Figures 1 and 2). N-(2,3-Epoxypropyl)phthalimide, 1,2-epoxy-3-phenoxypropane, and 2,3epoxypropyl p-methoxyphenyl ether were moderately cytotoxic in both strains and highly genotoxic in the

Activation and Inactivation of Carcinogens by GST 5-5

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 335

Figure 1. Induction of umu gene expression (1A, 2A, 3A, 4A, and 5A) and cytotoxicity response (1B, 2B, 3B, 4B, and 5B) by carcinogenic chemicals in tester TA1535/pSK1002 (0) and NM5004 (9) strains. The induction of umu gene expression is represented as β-galactosidase activity (A420 using o-nitrophenyl β-D-galactopyranoside as a substrate). Cytotoxicity activities are expressed as % of optical density change at 600 nm.

Figure 2. Induction of umu gene expression (6A, 7A, 8A, 9A, and 10A) and cytotoxicity response (6B, 7B, 8B, 9B, and 10B) by carcinogenic chemicals in tester TA1535/pSK1002 (0) and NM5004 (9) strains. Other details are as in the legend to Figure 1.

NM5004 strain. Thus, the above 10 chemicals appear to be activated to genotoxic GSH conjugates in the NM5004 tester strain. In contrast, ENPP was inactivated by GST activity in the NM5004 strain because it induced umu gene expression in the original TA1535/pSK1002 strain but not in the NM5004 strain, although this chemical was cytotoxic in both tester strains (Figure 3). 2,3-Dibromo-1-chloropropane was slightly more genotoxic in the TA1535/ pSK1002 than in the NM5004 strain; the chemical was moderately cytotoxic in both strains. These chemicals appear to be inactivated in the NM5004 strain. Although 1,4-dibromo-2,3-epoxybutane was highly genotoxic in the NM5004 strain, this chemical was more cytotoxic in the NM5004 strain than TA1535/pSK1002 (Figure 3).

Using this approach, we compared the abilities of 29 chemicals to induce umu gene expression and to cause cytotoxicity in two tester strains, in order to determine whether these chemicals are activated or inactivated by GST activity (Table 1). Potencies of chemicals that induced genotoxicity and cytotoxicity in two tester strains were ranked as described in the legend to Table 1. We also compared the present umu results with the mutagenic activities of several of chemicals that were reported using Ames tester strains (8, 11). As described above, 10 of the chemicals s1,2-dibromoethane, N-(2,3epoxypropyl)phthalimide, 1,3-dichloroacetone, CH2I2, 1,2epoxy-3-phenoxypropane, 2,3-epoxypropyl p-methoxyphenyl ether, 1-bromo-2-chloroethane, 1-bromo-2,3-dichloropropane, CH2BrCl, and CH2Br2sappear to be activated


Shimada et al.

Table 1. Comparison between umu and Ames Test Systems To Induce Genotoxicity Response and Reverse Mutation by Carcinogenic Compoundsa umu test systemb

(1) 1,2-dibromoethane (2) N-(2,3-epoxypropyl)phthalimide (3) 1,3-dichloroacetone (4) CH2I2 (5) 1,2-epoxy-3-phenoxypropane (6) 2,3-epoxypropyl p-methoxyphenyl ether (7) 1-bromo-2-chloroethane (8) 1-bromo-2,3-dichloropropane (9) CH2BrCl (10) CH2Br2 (11) 1,2-epoxy-3-(4′-nitrophenoxy)propane (12) 2,3-dibromo-1-chloropropane (13) 1,4-dibromo-2,3-epoxybutane (14) 1,2-epoxy-3-bromopropane (15) 1,2-epoxy-3-chloropropane (16) 1,2,3,4-diepoxybutane (17) 2,3-dibromopropionaldehyde (18) 1,4-dibromo-2,3-dihydroxybutane (19) 1,4-dibromobutane (20) 1,3-dibromoacetone (21) 2,3-dibromo-1-propanol (22) 1,2-epoxy-4-bromobutane (23) CH2Cl2 (24) 1,3-dibromo-2-propanol (25) 1-bromo-2,3-propanediol (26) 4-vinylcyclohexene dioxide (27) cyclohexene oxide (28) 1,2-epoxybutane (29) 1-bromo-2-fluoroethane

conc

GST(+) umu tox

GST(-) umu tox

0.24 1.0 0.024 1.0 1.0 1.0 0.5 0.5 1.0 1.0 1.0 1.0 0.25 3.0 3.0 3.0 0.12 1.0 1.0 0.24 1.0 1.0 1.0 1.0 1.0 0.25 0.25 1.0 1.0

+++ +++ ++ ++ + + + + + + ( + + + ( + ( ( ( -

+ + ++ + ++ + ( + + + ( ( ( -

+++ ++ +++ + +++ +++ ++ ++ ++ +++ +++ + +++ +++ ( + +++ ( ++ ( -

++ +++ + +++ ++ ( ++ ++ + ++ ++ ++ ( ++ +++ ( ++ ( -

conc

Ames test systemc (revertants) GST(+) GST(-)

similarity: umu/Ames

1.0

+++

-

yes

0.05

+++

+

yes

2.0 1.0 0.2 0.6 0.01 0.2

+ ++ ( ( + +

++ (

yes yes yes ? ? ?

0.6 5.0 2.0 0.1

+++ ( ++ -

( + -

no yes yes yes

0.1 2.0 2.0 0.6 1.0

+ + ( + (

( ( ( (

? ? ? ? yes

+

(

?

10

a Potencies of chemicals in umu (genotoxicity and cytotoxicity) and Ames (mutagenicity) systems were ranked as follows: (-) 0-50; (() 50-100; (+) 100-250; (++) 250-450; (+++) 450 for for umu expression (units); (-) 90-100; (() 80-90; (+) 65-80; (++) 50-65; (+++) 50 for umu cytotoxicity (%); (-) 0-40; (() 40-80; (+) 80-200; (++) 200-400; (+++) 400 for Ames mutagenicity (revertants). b Present results. c Data from Their et al. (8, 11). Conc, maximum concentrations (mM) tested; umu, umu units/mL; tox, cytotoxicity (%); similarity, comparison with umu and Ames test systems.

Figure 3. Induction of umu gene expression (11A, 12A, and 13A) and cytotoxicity response (11B, 12B, 13B) by carcinogenic chemicals in tester TA1535/pSK1002 (0) and NM5004 (9) strains. Other details are as in the legend to Figure 1.

by GST 5-5. Two chemicalssENPP and 2,3-dibromo-1chloropropanesappear to be inactivated by GST 5-5. Some of the other chemicals showed different responses for genotoxicity (as measured by β-galactosidase activities) and cytotoxicity (as measured by absorbance change at 600 nm) (Table 1). These chemicalss1,4-dibromo-2,3-

epoxybutane, 1,2-epoxy-3-bromopropane, 1,2-epoxy-3chloropropane, 1,2,3,4-diepoxybutane, 2,3-dibromopropionaldehyde, 1,4-dibromo-2,3-dihydroxybutane, and 1,4dibromobutanesgave different responses in the two tester strains, but we could not define whether these chemicals are activated or inactivated by GST 5-5. For example, 1,4-dibromo-2,3-epoxybutane was found to be less genotoxic in the NM5004 strain but more cytotoxic in this strain than in TA1535/pSK1002 (Figure 3, Table 1). Similar results were obtained when 1,2-epoxy-3bromopropane and 1,2-epoxy-3-chloropropane were tested. The other four chemicals of these sevens1,2,3,4-diepoxybutane, 2,3-dibromopropionaldehyde, 1,4-dibromo2,3-dihydroxybutane, and 1,4-dibromobutaneswere not very different in genotoxic responses in two strains but did show different patterns in cytotoxic responses. Several of the chemicals showed similar or no genotoxic and cytotoxic responses in the two tester strains. This group included 1,3-dibromoacetone, 2,3-dibromo-1-propanol, 1,2-epoxy-4-bromobutane, CH2Cl2, 1,3-dibromo-2propanol, 1-bromo-2,3-propanediol, 4-vinylcyclohexene dioxide, cyclohexene oxide, 1,2-epoxybutane, and 1-bromo2-fluoroethane (Table 1). Evidence for Involvement of GST 5-5 in the Inactivation of Reactive Metabolites of Procarcinogens Formed by Liver Microsomes of PCBTreated Rats. Several procarcinogens were oxidized by liver microsomes of PCB-treated rats in the presence of tester bacterial strains in order to see whether or not the genotoxicity of the products differed in the two tester strains. Dependence of induction of umu gene expression


Figure 4. Dependence of induction of umu gene expression on concentrations of microsomal proteins (A and B) and carcinogens (C and D) in TA1535/pSK1002 (A and C) and NM5004 (B and D) after oxidation of MeIQ (b), (+)B[a]P-7,8-diol (4), sterigmatocystin (O), and AFB1 (2) by rat liver microsomes. Table 2. Activation of Procarcinogens by Liver Microsomes of PCB-Treated Rats in S. typhimurium TA1535/pSK1002 and NM5004a umu gene expression [units min-1 (mg of protein)-1] MeIQ MeIQx IQ Trp-P-1 6-AC 6-AC-diol AFB1 sterigmatocystin (+)-7,8-B[a]P-diol (-)-7,8-B[a]P-diol

TA1535/pSK1002 (A)

NM5004 (B)

A/B

1869 497 576 355 3076 470 598 707 1538 905

1846 428 385 155 1406 28 22 29 97 52

1.0 1.2 1.5 2.3 2.2 17 27 24 16 17

a Procarcinogens (5 µM in all cases except where 1.0 µM 6-aminochrysene was used) were incubated with liver microsomes (5 µg of protein/mL of incubation) from polychlorinated biphenyltreated rats in the presence of tester bacterial strains, and induction of umu gene expression was determined as described in the Experimental Procedures. Determinations were conducted in triplicate; the standard deviations in these assays were less than 15% of the mean values.

on concentrations of microsomal protein and procarcinogens was determined (Figure 4). The induction of umu gene expression by 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ), trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(+)B[a]P-7,8-diol], sterigmatocystin, and AFB1 was increased after activation by rat liver microsomes in the TA1535/pSK1002 strain. MeIQ showed similar responses in NM5004 as with the TA1535/pSK1002 strain, but the other three compounds showed extremely low induction of umu gene expression in the NM5004 tester strain (Figure 4). We also compared the activation of several other procarcinogens by liver microsomes of PCB-treated rats in the two bacterial strains (Table 2). MeIQ, 2-amino3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 6-aminochrysene (6-AC) were found to be genotoxic in both


tester strains after metabolic activation by liver microsomes. However, other procarcinogens including trans1,2-dihydro-1,2-dihydroxy-6-aminochrysene (6-AC-diol), AFB1, sterigmatocystin, and the (+)- and (-)-enantiomers of B[a]P-7,8-diol were highly genotoxic only in the TA1535/pSK1002 strain; the ratios of induction of umu gene expression between the TA1535/pSK1002 and the NM5004 strain were >15 with these chemicals. HPLC Analysis of AFB1 Metabolites Formed by Liver Microsomes of PCB-Treated Rats and by Human Liver Microsomes. The above results suggested that epoxide intermediates of several procarcinogens may be trapped by GST 5-5 to form inactive GSH conjugates in the NM5004 strain. To test this hypothesis, we analyzed GSH conjugates and other metabolites of AFB1 after oxidation by liver microsomes of PCBtreated rats or by human liver microsomes in the presence of a purified rat GST mixture (Sigma) or GST Yrs-Yrs (a theta class rat GST enzyme) for detecting GSH conjugates of AFB1 after metabolic activation. Without addition of GSH, rat liver microsomes mediated significant formation of AFM1 and AFQ1, but not AFB1-8,9-epoxide-GSH conjugates, in the presence of purified rat GSTs and GST Yrs-Yrs (Figure 5A,B). With the addition of 3 mM GSH to the reaction mixtures, the purified rat GST and GST Yrs-Yrs gave significant amounts of AFB1-8,9-epoxide-GSH conjugate (Figure 5E,F). Microsomes of liver sample HL-4 (with a high amount of P450 3A4) produced high amounts of AFQ1 (but not AFM1) after incubation of AFB1 in the presence of purified rat GST or GST Yrs-Yrs (without GSH) (Figure 5C,D). AFB1-8,9-epoxide-GSH adducts were detected in systems containing both purified rat GSTs or GST Yrs-Yrs only in the presence of GSH (Figure 5D,F).

Discussion Thier et al. (8) constructed a new tester Salmonella strain by introducing rat GST 5-5 cDNA into S. typhimurium TA1535 and found that this tester strain can activate dihaloalkanes to mutagenic products. Similar approaches by Wolf and his associates were also carried out by introducing human GST cDNAs (alpha and pi class GSTs) into S. typhimurium TA1535 and TA100 strains to detect the mutagenic potential of environmental carcinogens (9, 10). Recently, Thier et al. (11) have extended the work on mechanisms of GST 5-5-dependent activation of several bifunctional alkylating agents, using their tester strain for the detection of mutagenic activities, and postulated mechanisms involving episulfonium ions in the mutagenic activation of these compounds by GST 5-5. Using an original tester strainsS. typhimurium TA1535/pSK1002sand a newly developed tester strainsNM5004, in which a plasmid containing both rat GST 5-5 cDNA and the umuC′′lacZ operon had been introduced into the host strain S. typhimurium TA1535swe compared 29 chemicals that were suggested to be conjugated by GST in terms of their abilities to induce umu gene expression and cytotoxicity responses in these tester strains. Ten of the 29 were found to be more genotoxic in the NM5004 strain than in the TA1535/pSK1002 strain and thus appear to be activated to form reactive GSH conjugates by GST 5-5. These chemicals include 1,2-dibromoethane, N-(2,3-epoxypropyl)phthalimide, 1,3-dichloroacetone, CH2I2, 1,2-epoxy-3phenoxypropane, 2,3-epoxypropyl p-methoxyphenyl ether,


Shimada et al.

Figure 5. High-performance liquid chromatography of GSH-conjugates and other metabolites of AFB1 after oxidation by rat (A, B, E, and F) and human (C, D, G, and H) liver microsomes in the presence of rat GSTs (A, C, E, and G)), and GST Yrs-Yrs (B, D, F, and H). GSH (3 mM) was added in E, F, G, and H. Peaks a, c, d, and e were identified as GSH conjugates of exo-AFB1-epoxide and AFQ1, AFM1, and AFB1, respectively. Peak b corresponds to residual AFB1-8,9-dihydrodiol (31).

Figure 6. Proposed pathways for activation of several chemicals by GST 5-5. Chemical names are as follows: (A) 1,2dibromoethane and 1-bromo-2-chloroethane; (B) 1-bromo-2,3dichloropropane; (C) N-(2,3-epoxypropyl)phthalimide; (D) 1,2epoxy-3-phenoxypropane; (E) 2,3-epoxypropyl p-methoxyphenyl ether; and (F) CH2I2, CH2BrCl, and CH2Br2.

1-bromo-2-chloroethane, 1-bromo-2,3-dichloropropane, CH2BrCl, and CH2Br2. Of these chemicals, 1,2-dibromoethane, N-(2,3-epoxypropyl)phthalimide, 1,2-epoxy-3phenoxypropane, 2,3-epoxypropyl p-methoxyphenyl ether, 1-bromo-2-chloroethane, and 1-bromo-2,3-dichloropropane are postulated to be converted to episulfonium ions by GST 5-5 (Figure 6). The dihalomethanes CH2I2, CH2BrCl, and CH2Br2 are activated by GST 5-5 through formation of S-(1-haloalkyl)GSH products (8). However, detailed information is lacking regarding the activation of 1,3-dichloroacetone by GST 5-5 (8, 19, 20). 1,4-Dibromo-2,3-epoxybutane, 1,2-epoxy-4-bromobutane, and 1,2,3,4-diepoxybutane have been reported to show enhanced (base-pair) mutagenicity in an S. typh-

imurium TA1535 (+GST) strain (11). Mechanisms involving the formation of episulfonium ions have been proposed in the activation process, in contrast to 5-membered thialonium ions (11). The present results indicate that these chemicals were less genotoxic in the GSTexpressing strain in this system. It should be mentioned that these chemicals were highly cytotoxic in both tester strains in the SOS response system and also that it is sometimes difficult to determine the cytotoxic responses of chemicals in the Ames test system. Different end points of genotoxicity may also explain different responses in the Ames and umu test systems. The Ames test results (TA1535 system) are dependent on the ability of (activated) chemicals to bind to a single pair of GC pairs and cause mutations that yield his+ revertants (35, 36). The umu response is based upon the induction of the SOS response, due to blockage of DNA polymerase and changes in nucleotide pools (26, 27). Many of the compounds under consideration here are bifunctional alkylating agents and are likely to produce cross-links. Crosslinks might tend to favor SOS response more than base-pair substitutions. Although there is a generally good correlation between the responses of chemicals in the umu and reversion assay (26, 37), it is not surprising that some distinctions will be seen. It is also of interest to note that 1,2,3,4-diepoxybutane shows higher bacterial mutagenicity in the presence of a GST system (11) but a lower incidence of sister chromatid exchange in human lymphocytes that express GST T1 (38). In this regard, the umu system seems to be better correlated than the Ames test. Although 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3bromopropane, 1,2-epoxy-3-chloropropane, 1,2,3,4-diepoxybutane, 2,3-didromopropionaldehyde, 1,4-dibromo2,3-dihydroxybutane, and 1,4-dibromobutane gave different responses in two tester strains, we could not determine whether these chemicals are activated or inactivated by GST 5-5. For example, 1,4-dibromo-2,3epoxybutane was found to be less genotoxic in the NM5004 strain but was more cytotoxic in this strain


(than in TA1535/pSK1002) (Table 1). However, ENPP appears to be inactivated in the NM5004 strain, with regard to its ability to induce umu gene expression, though it has cytotoxic activities. Inactivation of ENPP by an Ames tester strain expressing GST 5-5 has also been reported (11). However, mechanisms underlying inactivation of ENPP by GST 5-5 have not been elucidated yet. 2,3-Dibromo-1-chloropropane was also suggested in this study to be inactivated in the NM5004 strain. A number of studies have established that many chemical promutagens and procarcinogens require activation by P450 enzymes to form reactive metabolites that bind covalently to intracellular macromolecules (39, 40). These reactive metabolites are also conjugated with intracellular nucleophiles by Phase II enzymes such as GST and sulfotransferase to form inactive polar metabolites (7, 41, 42). Risks of these mutagens and carcinogens may be determined by the balance between activation by P450 enzymes and inactivation by Phase II enzymes (16, 43, 44). Several procarcinogens including AFB1, sterigmatocystin, 6-AC-diol, and the (+)- and (-)-enantiomers of B[a]P-7,8-diol showed less genotoxicity in the GSH 5-5-expressing NM5004 strain (after activation by liver microsomes of PCB-treated rats), suggesting that the active (epoxide) metabolites are trapped as polar GSH conjugates by the action of GST 5-5. This GST enzyme seems to be inactive toward the N-hydroxylated products of carcinogenic arylamines, because similar genotoxicity activities (umu gene expression) were determined in both tester strains after activation of MeIQ, MeIQx, IQ, TrpP-1, and 6-AC by rat liver microsomes. Inactivation of AFB1 by alpha and pi class human GST enzymes has also been suggested by Simula et al. (9). HPLC analysis of metabolites of AFB1 after oxidation by rat and human liver microsomes suggested that AFB18,9-epoxide-GSH conjugates could be formed when purified rat GSTs or rat theta class GST Yrs-Yrs were added with GSH (Figure 5). This is due to the action of GST activities, because in the absence of GSH, very little or trace amounts of GSH conjugates were determined with the above purified GST enzymes. Alpha and mu class GST enzymes have been reported to catalyze conjugation of AFB1 and benzo[a]pyrene oxidation products to form respective GSH conjugates (9, 44-46). However, it is not known that theta class GST enzymes have roles of these potential procarcinogens. The results that purified theta class GST Yrs-Yrs enzyme had significant (but not high) activities to form AFB1-8,9epoxide-GSH adducts are the first evidence to support the roles of this class enzyme to participate toward these potential carcinogens as well as dihaloalkanes and related carcinogens (8, 11). Recent studies have demonstrated that human theta class GSTT1, which shows 82% identity in nucleotide sequence with rat theta class GST 5-5, shows genetic polymorphism in humans; about 40% of human populations are defective in the expression of this GST gene (12). Genetic polymorphism of other GST genes including human mu GST as well as theta class GSTT1 has been reported, and some results suggest that mu class genetic polymorphism may be related to lung cancer in humans (47-51). Thus, the importance of understanding the basis for genetic polymorphism of these GST genes is increased, in order to determine the different susceptibilities of humans toward a number of environmental


chemicals that are mutagenic and potentially carcinogenic.

Acknowledgment. This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, the Ministry of Health and Welfare of Japan, and the Osaka Prefectural Government and by United States Public Health Service Grants CA44353 and ES00267.

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