German Institute of Human Nutrition (DIfE) Potsdam-Rehbruecke, Department of Toxicology, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany
1 To whom correspondence should be addressed Email: glatt{at}mail.dife.de
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Abstract |
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Abbreviations: NAT, acetyltransferase; NR, nitroreductase; OAT, endogenous acetyltransferase of Salmonella typhimurium; SULT, sulfotransferase
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Introduction |
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The use of nitrofen as a herbicide was prohibited in most countries a long time ago, in the Federal Republic of Germany in 1988, and in its new states (Eastern Germany) in 1990. Nevertheless, a substantial contamination of organically produced chicken occurred in summer 2002 in Germany through storage of feed in a warehouse previously used for storing pesticides (8). This event renewed interest in the toxicological effects of nitrofen and the underlying mechanisms.
Mutagenicity is a major mechanism of chemical carcinogenicity; in general, it involves the covalent binding of a reactive species to DNA. Reactive species are also an established cause of teratogenic effects. Nitrofen is a nitroarene. Numerous nitro- and aminoarenes are metabolically activated to mutagens in two steps: formation of the corresponding hydroxylamine by reduction and oxidation, respectively, and subsequent conjugation, usually by acetyltransferases (NATs) or sulfotransferases (SULTs), to generate a reactive ester (911). It has been shown that nitro reduction is an important metabolic pathway of nitrofen in animal models; in addition, ring hydroxylation has been observed (1214).
The results of short-term mutagenicity assays with nitrofen were usually negative, weakly positive or controversial. Most data were obtained with the reversion assay with his Salmonella typhimurium strains. In the first study, nitrofen was mutagenic in strain TA98 in the presence and absence of liver post- mitochondrial preparations (S9) from Aroclor 1254-treated rats and in strain TA100 only in the presence of S9 (15). Moriya et al. (16) found similar mutagenicity in strain TA100 in the presence and absence of S9, whereas effects in TA98 were detected only in its presence. Shirasu et al. (17) observed mutagenicity of nitrofen in strain TA100 in the presence and absence of S9, whereas Draper and Casida (13) reported a negative result under the same conditions. A possible explanation for these conflicting results was provided by Seiler (18). He observed that a contaminant, bis(4-nitrophenyl)ether, caused the mutagenic effects observed with technical nitrofen preparations, whereas purified nitrofen was negative under the conditions used (strain TA100 in the absence of a liver enzyme preparation). In a study of Tanaka et al. (19) nitrofen (tested in the presence of liver S9) was inactive in strain TA100, but highly mutagenic in a TA100-derived strain engineered for over-expression of an endogenous nitroreductase (YG1026) and moderately mutagenic in another TA100-derived strain over-expressing an endogenous acetyltransferase (YG1029). Several reduced derivatives of nitrofen were clearly mutagenic, amino-nitrofen only in the presence of S9, nitroso- and hydroxylamino-nitrofen in the presence and absence of S9 (13,19,20). The mutagenicity of amino-nitrofen was strongly enhanced in strains over-expressing acetyltransferase, but unaffected in those over-expressing nitroreductase compared with control strains TA100 and TA98 (19). These various results indicate that nitrofen may be activated to a mutagen via nitro reduction and subsequent acetylation. Endogenous enzymes of Salmonella are able to catalyze these reactions, at least if they are strongly over-expressed. However, the catalytic activities of Salmonella are of minor interest with regard to the risk for humans. This leads to the question whether corresponding activities occur in the mammalian organism. Nitro reduction has been found to be a major metabolic pathway of nitrofen in various animal models (1214). However, nothing is known on the subsequent esterification reaction of the hydroxylamine by human enzymes in particular and by mammalian enzymes in general. Two human acetyltransferases (NAT1 and NAT2) have been detected that can activate aromatic hydroxylamines (21,22). Sulfonation is another conjugation reaction shown to be important as the final activation step of many nitro- and aminoarenes (23,24). Sulfonation of xenobiotics is mediated by soluble enzymes of the SULT superfamily (25,26). A total of 11 SULT forms have been detected in humans (26,27). It has been shown that several human SULT forms can activate aromatic hydroxylamines (28). Moreover, since the substrate specificity of the remaining forms has not been sufficiently explored, it cannot be ruled out that they catalyze the activation of other aromatic hydroxylamines. It is interesting to identify all human SULTs involved in the activation of a given promutagen/procarcinogen, since SULTs strongly differ in their tissue distribution, and some forms are genetically polymorphic (26). Identification of the forms activating a given compound may allow creating educated hypotheses on potential target tissues and individual susceptibility.
The aim of this study was to identify mammalian, in particular human, SULTs and NATs that are capable of mediating the activation of nitrofen. Reactive sulfuric acid esters do not readily permeate into bacteria, as they are ionized species (29). For this reason, we have expressed the enzymes directly in Salmonella target strains. In particular, we expressed all human SULT forms identified in S.typhimurium TA1538 (28) and most forms also in strain TA100. Human NATs were the first mammalian xenobiotic metabolizing to be expressed in S.typhimurium strains (30). They were expressed in a TA1538-derived strain deficient in the endogenous acetyltransferase of Salmonella (TA1538/1,8-DNP). In the present study, we used analogous strains constructed in our laboratory (31). In addition, we conducted some experiments using strains expressing important SULT forms from laboratory animal species and strains with altered expression of endogenous acetyltransferase or nitroreductase. Since all previous studies suggest that activation of nitrofen occurs at the nitro group, we did not use any S9 preparation.
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Materials and methods |
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Bacterial strains
Salmonella typhimurium strains TA1538, TA98 and TA100 were kindly provided by Dr B.N.Ames (Berkeley, CA). TA1538 and TA98 have lost their ability to synthesize histidine through a 1 frameshift mutation and are sensitive to frameshift mutagens (32). TA100 contains a substitution mutation in the histidine operon and can be reverted by several different substitution mutations and in-frame deletions (32). TA98 and TA100but not TA1538contain the plasmid pKM101, which encodes an error-prone repair system and an ampicillin resistance marker.
Salmonella typhimurium strains YG7131 and YG1024 were generous gifts of Dr T.Nohmi (National Institute of Health Sciences, Tokyo). Strain YG7131, derived from TA1538, lacks nitroreductase activity due to targeted disruption of the gene encoding the classical nitroreductase and natural deficiency of another nitroreductase (33). Gene disruption involved the integration of a kanamycin resistance marker. Strain YG1024 is derived from TA98 via insertion of a plasmid that encodes the endogenous acetyltransferase OAT (34); this modification results in a nearly 100-fold increase in acetyltransferase activity compared with the parental strain.
Salmonella typhimurium strain TA1538/1,8-DNP was kindly provided by Dr D.Wild (Bundesanstalt für Fleischforschung, Kulmbach, Germany). McCoy et al. (35) selected a TA98-derived mutant that was resistant towards 1,8-dinitropyrene. This strain, TA98/1,8-DNP6, lacks OAT activity (35). Watanabe et al. (34) eliminated plasmid pKM101 from this strain to generate TA1538/1,8-DNP.
The cloning of human SULT cDNAs into vector pKK233-2 (encoding an ampicillin resistance marker) and its expression in S.typhimurium TA1538 has been described in previous publications (28,36,37). In all cases, the reference (wild-type) amino acid sequence was expressed (GenBank accession numbers in ref. 28), although in some cases silent nucleotide exchanges were introduced for various technical reasons into some human cDNAs (28). Three rodent cDNAs expressed were synonymous with published sequences, rat (r) SULT1A1 (GenBank accession number X52883), rSULT1C1 (L22339) and mSULT1C2 (AY005469). The mSULT1A1 cDNA isolated in our laboratory (from an NMRI mouse) shows four non-synonymous exchanges compared with sequence L02331 (Asn55Thr, Ser171Pro, Tyr256Ser and Leu286Ile); we have verified the correctness of our sequence (W.Meinl and H.R.Glatt, manuscript in preparation). pKK233-2-based vectors were used for the transformation of strains TA1538 and YG7131. For the expression of SULTs in strains TA100 and TA98, which already are ampicillin-resistant, the ampicillin resistance marker of pKK233-2 was replaced by a neomycin/kanamycin selection marker as follows. The cDNA of the neomycin resistance marker contained in the plasmid pNeo (Amersham Pharmacia Biotech, Little Chalfont, UK) was excised with BamHI and HindIII, subsequently blunted using the large fragment of Escherichia coli DNA polymerase I and then ligated into the blunted, unique PvuI site located approximately in the middle of the ampicillin resistance sequence of pKK233-2 to yield a vector designated pKKneo. pKK233-2-SULT vectors were digested using SalI and HindIII restriction enzymes. The SULT-containing fragment was ligated into the pKKneo plasmid after removal of the corresponding 571 bp SalI- and HindIII-digested fragment.
Human NAT1 and NAT2 were expressed in TA1538/1,8-DNP using vector pKK233-2 (31). The resulting strains were similar to the corresponding strains constructed previously by Grant et al. (30).
The names of the recombinant strains are composed of the recipient strain and the expressed enzyme. In the present paper, no prefix is used for human forms, whereas r and m indicate rat and mouse enzymes.
Expression levels in the recombinant strains
Using purified enzyme protein as a standard, the expression levels could be estimated in some strains by immunoblotting. SULT1A1 constitutes 0.71.4% of the cytosolic protein of strain TA1538-SULT1A1 (28). The level in strain TA1538/1,8-DNP-SULT1A1 is similar to that in TA1538-SULT1A1, whereas the level found in TA100-SULT1A1 is lower by a factor of nearly two. The level in TA1538-SULT1A1 is nearly five times that observed in a liver sample studied concurrently. As estimated by immunoblotting, the level of SULT1A2 (in strain TA1538-SULT1A2*1Z) is 24% of the cytosolic protein (28).
No purified standards were available for most other SULTs. However, after electrophoresis of cytosolic preparations on polyacrylamide gels under denaturing conditions and Coomassie blue staining, an additional protein band (which also reacted with the appropriate anti-SULT antibody) was detected in each recombinant strain. We estimate that the intensity of the additional band varied from 6-fold weaker to 3-fold stronger compared with SULT1A1 in strain TA1538-SULT1A1.
Using an antibody raised against NAT1, strong signals were detected in cytosol preparations of strains TA1538/1,8-DNP-NAT1 and -NAT2 (31). Whereas the intensity of the immunosignals were similar, the electrophoretic mobility was different between the strains (31). Corresponding bands were also detected in eight cytosolic liver samples studied concurrently. However, hepatic levels were <1% of those observed in the recombinant strains.
Mutagenicity assay
The bacteria were grown in Nutrient Broth No. 2 (Oxoid GmbH, Wesel, Germany) at 37°C with shaking for 8 h. The parental strains TA1538, TA98 and TA100 were grown in the absence of antibiotics. Ampicillin (100 µg/ml) and neomycin (100 µg/ml) were added to the growth medium for the recombinant strains containing pKK233-2- and pKKneo-derived plasmids, respectively. The cultures were centrifuged, suspended in medium A (1.6 g/l Bacto Nutrient Broth + 5 g/l NaCl), adjusted nephelometrically to a titer of 12 x 109 bacteria (colony-forming units)/ml and kept on ice. Shortly before use they were centrifuged again and suspended at a 5-fold higher density in medium A.
Mutagenicity was determined using a modified version of the liquid- pre-incubation assay described by Maron and Ames (32). The bacterial suspension (100 µl) and the test compound (in 10 µl dimethylsulfoxide) were added sequentially to a glass tube containing 500 µl of 100 mM MgSO4. After incubation for 60 min at 37°C, 2.0 ml of 45°C warm soft agar (5.5 mg/ml agar, 5.5 mg/ml NaCl, 50 µM biotin, 50 µM histidine, 25 mM sodium phosphate buffer, pH 7.4) was added, and the mixture was poured onto a Petri dish containing 24 ml minimal agar (15 mg/ml agar in Vogel-Bonner E medium with 20 mg/ml glucose). After incubation for 3 days (TA1538/1,8-DNP) or 2 days (all other strains) in the dark, the colonies (his+ revertants) were counted. Incubations were carried out in triplicate.
Taking the previous mutagenicity studies with nitrofen as a guide, doses of 1, 3, 10, 30, 100, 300 and 1000 nmol of nitrofen were used in initial experiments with selected strains. In subsequent experiments, the factor between neighboring doses was reduced to 2 and the range was adjusted to the effects observed. In particular, lower dose levels were then used with strains expressing human SULT1A1. With non-responsive strains, the highest dose level was set to 2048 nmol (a level associated with some precipitation of test compound).
The result was evaluated as follows. It was classified positive, if the number of revertants (mean value at any dose level) was increased at least 2-fold above the number of spontaneous revertant colonies with a plausible doseresponse relationship. It was also classified positive if the increase was at least 1.5-fold and confirmed in a repeat experiment.
Specific mutagenicities (revertants per nmol) were calculated from the slope of the initial part of the doseresponse curve of the positive results. For negative results, a conservative limit of detection is given by dividing the number of spontaneous revertants by the highest dose that could be adequately tested (no obvious toxicity and no massive precipitation of test compound).
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Results |
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Influence of nitroreductase deficiency on SULT1A1-mediated mutagenicity
Nitroarenes have not been found to be substrates of SULTs; however, their reduction products, hydroxylamines and amines, may be conjugated (26). Indeed, reduction of nitrofen and other chlorinated 4-nitrobiphenyl ethers has been observed in S.typhimurium (20). Therefore, we suspected that nitrofen was reduced to the corresponding hydroxylamine before it was activated to a mutagen by human SULTs expressed in S.typhimurium. To test this hypothesis, we expressed human SULT1A1 in a TA1538-derived strain (YG7131) deficient in nitroreductases. The level of SULT1A1 expression was similar in strains TA1538-SULT1A1 and YG7131-SULT1A1 (data not shown). In addition, we have used a positive control, 1-hydroxymethylpyrene, which requires SULT, but no additional enzymes, for its activation to a mutagen. Indeed, 1-hydroxymethylpyrene showed similar mutagenic effects in strains YG7131-SULT1A1 and TA1538-SULT1A1 (Figure 4). However, nitrofen was mutagenic only to strain TA1538-SULT1A1, but not to strain YG7131-SULT1A1 (Figure 4). Thus, both nitoreductase as well as SULT activity were required for the activation of this nitroarene.
Mutagenicity in strains expressing rodent SULTs
Nitrofen was inactive in a TA1538-derived strain expressing mouse SULT1A1, and showed effects close to the limit of detection if rat SULT1A1 was expressed in that strain (Table I). It induced consistentalthough weakincreases in the number of revertant colonies if rat or mouse SULT1A1 was expressed in TA100 (Table I). Nitrofen was inactive in strains expressing rat SULT1C1 or mouse SULT1C2 (Table I).
Role of acetyltransferases in the activation of nitrofen
Conventional strains of S.typhimurium express an endogenous acetyltransferase, termed OAT by some authors. Nitrofen was inactive in strains TA100, TA1538 and TA98 (see above), but demonstrated clear mutagenic effects in a TA98-derived strain (YG1024) that strongly over-expresses OAT (Figure 5). In subsequent investigations, we expressed the two human NATs identified (NAT1 and NAT2) in an OAT-deficient mutant strain derived from TA1538, termed TA1538/1,8-DNP. Nitrofen was not mutagenic in the starting strain TA1538/1,8-DNP nor in the corresponding strain expressing human NAT1, but was weakly mutagenic in strain TA1538/1,8-DNP-NAT2 (Figure 6).
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Discussion |
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The aim of this study was to find whether this characteristic activation pathway of nitroarenes also occurs with nitrofen and to identify conjugating enzymes that can mediate the second activation step.
We demonstrate that nitrofen is activated to a mutagen in S.typhimurium strains expressing certain mammalian SULTs, in particular human SULT1A1. This activation is dependent on the presence of a nitroreductase. Therefore, it is probable that nitrofen is reduced to the corresponding hydroxylamines and then conjugated to a reactive sulfuric acid ester.
SULT-dependent metabolites led to reversion of substitution-as well as frameshift-mutation-sensitive strains (TA100 and TA1538 recipient strains, respectively). This result is not unusual, since many other bioactivated nitro- and aminoarenes also show both activities (10). The fact that the maximal mutagenic response occurred at a lower nitrofen concentration in strain TA1538-SULT1A1 than in strain TA100-SULT1A1 might be due to the higher SULT expression level in the former strain and/or a pKM101-plasmid-mediated protection against bacteriotoxic effects of nitrofen or its metabolites in recipient strain TA100.
In addition, we observed a second activation mechanism involving acetylation. Such a pathway had already been reported using strains that over-express an endogenous acetyltransferase (OAT) (19). Employing the OAT-over-expressing strain YG1024, we confirmed this finding. We found that this effect occurs even in the absence of a liver S9 preparation. We then studied the mutagenicity of nitrofen in strains expressing human NATs. One human NAT (NAT2) demonstrated a weak activation of nitrofen, close to the limit of detection, and the other form (NAT1) did not show any effect. This was the case, although the expression of NAT1 and NAT2 in strains TA1538/1,8-DNP-NAT1 and -NAT2 is extremely high compared with that in human tissues (>100-fold above the highest hepatic level found among eight subjects). We suspect that SULTs, in particular SULT1A1, are much more important for the activation of nitrofen in humans than is NAT2, since the mutagenic activity in the SULT1A1-expressing strains was stronger and the SULT1A1 level in these strains only was moderately higher than that found in the liver samples studied.
SULT1A1 was more active than any of the other 10 human SULTs identified. Older names of SULT1A1 are phenol- preferring phenol sulfotransferase (P-PST) and thermostable sulfotransferase (TS PST). It is the major broad-spectrum phenol SULT in humans. Despite the name, its substrate specificity is not limited to phenols, but also includes benzylic alcohols and thiols, N-oxides (such as minoxidil) and aromatic amines, hydroxylamines and hydroxamic acids (26). It is highly expressed in liver and has been detected at lower levels in numerous extrahepatic tissues (26). The abundance of this enzyme suggests that humans have a high capacity to activate nitrofen to mutagenic metabolites.
We also have investigated the orthologous enzymes of rodent species that have been used in carcinogenicity studies with nitrofen, i.e. mouse and rat. Although rat and mouse SULT1A1 activated nitrofen, they were clearly less active than human SULT1A1. Other rodent SULTs investigated (rat SULT1C1 and murine SULT1C2) were inactive, whereas a human enzyme from the same subfamily (SULT1C1) led to reproducible but weak activation of nitrofen. With the limitation that we have only investigated a small number of rodent enzymes (which however include the mouse and rat orthologs of the most efficient human enzyme, SULT1A1), it appears that human SULTs have higher activity towards the hydroxylamine of nitrofen than rat and mouse SULTs. Therefore, humans might be more susceptible to the carcinogenic action of this herbicide than standard animal models. Of course, the toxifying enzyme is not the only possible susceptibility factor.
SULT expression and activities at prenatal stages have been studied primarily in the human. While no data appear to be available for the embryonic phase, SULT activities towards numerous substrates have been detected in many fetal tissues, even at the earliest stages investigated (4146). Especially in extrahepatic tissues (including adrenal gland, lung, kidney, gut), these activities are often many-fold higher than the corresponding activities in adult tissues. For example, Richard et al. (46) observed six, five and nine times higher SULT activities towards dopamine, 4-nitrophenol and 3,3'-diiodothyronine, respectively, in fetal lung compared with postnatal lung. The corresponding values for the liver amounted to 8, 1.2 and 1.1. SULT1C2 mRNA was detected in the lung and heart of the fetus but not of the adult, and its level was higher in fetal kidney than in adult kidney (47). SULT1C1 mRNA was detected in fetal, but not adult, liver (48). Likewise, the laboratory of M.Coughtrie observed SULT1C1 and SULT1C2 protein expression in various fetal tissues, but not in adult tissue (49). SULTs frequently compete with UDP- glucuronosyltransferases for the same substrates. However, UDP-glucuronosyltransferases tend to develop only late in ontogeny (42,44,5053). This differential ontogenetic development of the two classes of conjugating enzymes is still detected in the newborn, whose ratio of urinary sulfo and glucuronic acid conjugates (e.g. of ritodrine) is markedly higher than in adults (54). Thus, the fetus may be particularly sensitive towards protoxicants that are activated by SULT and detoxified by competing UDP-glucuronosyltransferases. It is tempting to link this enzymatic situation with the observations that nitrofen is a relatively potent teratogen (shown in rodents) and is toxified to a reactive/mutagenic metabolite by SULT. If the hypothesis that SULT is involved in the teratogenicity of nitrofen is correct, then humans may be considered to be a susceptible species. While this hypothesis should be pursued, one has to keep in mind that nitrofen does not lead to an excess of embryonic death nor to massive cytotoxicity in embryonic tissues (55). This finding may argue against a major role of reactive metabolites. Rather, evidence is accumulating that nitrofen exerts a teratogenic effect via alterations in thyroid hormone status (55). The situation is complicated by the fact that SULTs are important in the regulation of thyroid hormones (56,57). Thus, competition between nitrofen metabolites and iodothyronines for SULT enzymes could also be a factor in nitrofen teratogenesis, with or without a role of reactive sulfuric esters formed from nitrofen.
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Acknowledgments |
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References |
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