The influence of the SULT1A status - wild-type, knockout or humanized - on the DNA adduct formation by methyleugenol in extrahepatic tissues of mice.

Methyleugenol, present in herbs and spices, has demonstrated carcinogenic activity in the liver and, to a lesser extent, in extrahepatic tissues of rats and mice. It forms DNA adducts after hydroxylation and sulphation. As previously reported, hepatic DNA adduct formation by methyleugenol in mice is strongly affected by their sulphotransferase (SULT) 1A status. Now, we analysed the adduct formation in extrahepatic tissues. The time course of the adduct levels was determined in transgenic (tg) mice, expressing human SULT1A1/2, after oral administration of methyleugenol (50 mg per kg body mass). Nearly maximal adduct levels were observed 6 h after treatment. They followed the order: liver > caecum > kidney > colon > stomach > small intestine > lung > spleen. We then selected liver, caecum, kidney and stomach for the main study, in which four mouse lines [wild-type (wt), Sult1a1-knockout (ko), tg, and humanized (ko-tg)] were treated with methyleugenol at varying dose levels. In the liver, caecum and kidney, adduct formation was nearly completely dependent on the expression of SULT1A enzymes. In the liver, human SULT1A1/2 led to higher adduct levels than mouse Sult1a1, and the effects of both enzymes were approximately additive. In the caecum, human SULT1A1/2 and mouse Sult1a1 were nearly equally effective, again with additive effects in tg mice. In the kidney, only human SULT1A1/2 played a role: no adducts were detected in wt and ko mice even at the highest dose tested and the adduct levels were similar in tg and ko-tg mice. In the stomach, adduct formation was unaffected by the SULT1A status. IN CONCLUSION: (i) the SULT1A enzymes only affected adduct formation in those tissues in which they are highly expressed (mouse Sult1a1 in the liver and caecum, but not in the kidney and stomach; human SULT1A1/2 in the liver, caecum and kidney, not in the stomach of tg mice and humans), indicating a dominating role of local bioactivation; (ii) the additivity of the effects of both enzymes in the liver and caecum implies that the enzyme level was limiting in the adduct formation; (iii) SULT1A forms dominated the activation of methyleugenol in several tissues, but non-Sult1a1 forms or SULT-independent mechanisms were involved in its adduct formation in the stomach.

Fruits and vegetables protect against the genotoxicity of heterocyclic aromatic amines activated by human xenobiotic-metabolizing enzymes expressed in immortal mammalian cells.

Heterocyclic aromatic amines (HAAs) can be formed during the cooking of meat and fish at elevated temperatures and are associated with an increased risk for cancer. On the other hand, epidemiological findings suggest that foods rich in fruits and vegetables can protect against cancer. In the present study three teas, two wines, and the juices of 15 fruits and 11 vegetables were investigated for their protective effect against the genotoxic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). To closely mimic the enzymatic activation of these HAAs in humans, genetically engineered V79 Chinese hamster fibroblasts were employed that express human cytochrome P450-dependent monooxygenase (hCYP) 1A2 (responsible for the first step of enzymatic activation) and human N(O)-acetyltransferase (hNAT) 2*4 or human sulfotransferase (hSULT)1A1*1 (responsible for the second step of enzymatic activation): V79-hCYP1A2-hNAT2*4 for IQ activation and V79-hCYP1A2-hSULT1A1*1 for PhIP activation. HAA genotoxicity was determined by use of the comet assay. Black, green and rooibos tea moderately reduced the genotoxicity of IQ (IC(50)=0.8-0.9%), whereas red and white wine were less active. From the fruit juices, sweet cherry juice exhibited the highest inhibitory effect on IQ genotoxicity (IC(50)=0.17%), followed by juices from kiwi fruit, plum and blueberry (IC(50)=0.48-0.71%). The juices from watermelon, blackberry, strawberry, black currant, and Red delicious apple showed moderate suppression, whereas sour cherry, grapefruit, red currant, and pineapple juices were only weakly active. Granny Smith apple juice and orange juice proved inactive. Of the vegetable juices, strong inhibition of IQ genotoxicity was only seen with spinach and onion juices (IC(50)=0.42-0.54%). Broccoli, cauliflower, beetroot, sweet pepper, tomato, chard, and red-cabbage juices suppressed IQ genotoxicity only moderately, whereas cucumber juice was ineffective. In most cases, fruits and vegetables inhibited PhIP genotoxicity less strongly than IQ genotoxicity. As one possible mechanism of antigenotoxicity, the inhibition of activating enzymes was studied either indirectly with diagnostic substrates or directly by measuring CYP1A2 inhibition. Only sour cherry, blueberry, and black currant juices suppressed the first step of HAA enzymatic activation, whereas most plant-derived beverages inhibited the second step.

Dietary exposure to 5-hydroxymethylfurfural from Norwegian food and correlations with urine metabolites of short-term exposure.

5-Hydroxymethylfurfural (HMF) is formed in carbohydrate-rich food during acid-catalysed dehydration and in the Maillard reaction from reducing sugars. HMF is found in mg quantities per kg in various foods. HMF is mainly metabolised to 5-hydroxymethyl-2-furoic acid (HMFA), but unknown quantities of the mutagenic 5-sulphoxymethylfurfural (SMF) may also be formed, making HMF potentially hazardous to humans. We determined the HMF content in Norwegian food items and estimated the dietary intake of HMF in 53 volunteers by means of 24h dietary recall. The estimated intakes of HMF were correlated with urinary excretion of HMFA. Coffee, prunes, dark beer, canned peaches and raisins had the highest levels of HMF. The 95th percentile of the estimated daily dietary intake of HMF and the 24h urinary excretion of HMFA were 27.6 and 28.6mg, respectively. Coffee, dried fruit, honey and alcohol were identified as independent determinants of urinary HMFA excretion. Most participants had lower estimated HMF intake than the amount of HMFA excreted in urine. In spite of this there was a significant correlation (r=0.57, P<0.001) between the estimated HMF intake and urinary HMFA. Further studies are needed to reveal alternative sources for HMF exposure.

Phytoestrogens and xenoestrogens: the contribution of diet and environment to endocrine disruption.

Some endocrine disrupting compounds such as phthalates and phenols act non-genomically by inhibiting the sulfotransferase (SULT 1E1 and SULT 1A1) isoforms which inactivate estrogens by sulfonation. A range of environmental phenolic contaminants and dietary flavonoids was tested for inhibition of the human SULT 1A1, 1E1 and 2A1 isoforms. In particular, the plasticisers 4-n-octyl- and 4-n-nonyl-phenol inhibit SULT 1E1 with IC(50) values of 0.16 microM vs. 10nM estradiol while the 2-substituted chlorophenols show similar values. Flavonoids are also SULT inhibitors; tricin is a competitive inhibitor of SULT 1E1 with a K(i) of 1.5+/-0.8 nM. In a small pilot study to determine whether ingestion of soy flavonoids would affect SULT1A1 activity in vivo as well as in vitro, sulfonation of daidzein was reduced in a group of women 'at risk' of breast cancer, as compared with controls, although the SULT 1A1*1/SULT 1A1*2 allele ratio was not different. Endocrine disrupting effects in man may be multifactorial when components from both the diet and the environment act at the same point in steroid metabolism.

Coffee diterpenes prevent the genotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N-nitrosodimethylamine in a human derived liver cell line (HepG2).

Aim of the present experiments was to study the genotoxic effects of coffee diterpenoids, namely cafestol palmitate and a mix of cafestol and kahweol (C+K) in human derived hepatoma (HepG2) cells. Furthermore, we investigated the potential protective properties of these substances towards carcinogens contained in the human diet, namely N-nitrosodimethylamine (NDMA) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). C+K and cafestol palmitate were tested over a broad dose range in micronucleus (MN) assays and no indication for genotoxic effects was seen. In combination experiments with PhIP (300 microM), pronounced inhibition (approximately 1.7-fold) of MN formation was observed with C+K and cafestol palmitate at dose levels > or = 0.9 and 1.7 microg/ml, respectively. Enzyme measurements indicate that the protection is due to inhibition of sulfotransferase, an enzyme involved in the activation of the amine, and/or to induction of UDP-glucuronosyltransferase which detoxifies the DNA-reactive metabolites of PhIP. Furthermore, a significant increase of glutathione-S-transferase was seen, whereas the activities of cytochrome P-450 1A1 and N-acetyltransferase 1 were not significantly altered. Also in combination experiments with C+K and NDMA, strong protective effects (50% reduction of genotoxicity) were seen at low dose levels (> or = 0.3 microg/ml). Since inhibition of MN was also observed when C+K were added after incubation with NDMA, it is likely that the chemoprotective effects are due to induction of DNA repair enzymes. Comparison of data on the effects of C+K on the cholesterol metabolism, which was investigated in earlier in vivo studies, with the present findings suggests that DNA-protective effects take place at exposure levels which are substantially lower than those which cause hypercholesterolemia.

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells.

Aim of the present study was the investigation of the genotoxicity of amino-alpha-carboline (AalphaC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AalphaC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations > or =50 microM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AalphaC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 microM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at > or =50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AalphaC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AalphaC is higher than that of other HAs that are formed during the cooking of meat and fish.

Protection by beverages, fruits, vegetables, herbs, and flavonoids against genotoxicity of 2-acetylaminofluorene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in metabolically competent V79 cells.

Chinese hamster lung fibroblasts, genetically engineered for the expression of rat cytochrome P450 dependent monooxygenase 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2-rSULT1C1 cells), were utilized to check for possible protective effects of beverages of plant origin, fruits, vegetables, and spices against genotoxicity induced by 2-acetylaminofluorene (AAF) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Antigenotoxic activities of juices from spinach and red beets against AAF could be monitored with similar effectivity by the HPRT-mutagenicity test (IC(50)=0.64%; 2.57%) and alkaline single cell gel electrophoresis (comet assay; IC(50)=0.12%; 0.89%) which detects DNA strand breaks and abasic sites. Applying the comet assay, genotoxicity of PhIP could, however, be demonstrated only in the presence of hydroxyurea and 1-[beta-D-arabinofuranosyl]cytosine, known inhibitors of DNA repair synthesis. As expected, AAF and PhIP were unable to induce any genotoxic effects in the parent V79 cells. Genotoxic activity of PhIP was strongly reduced in a dose-related manner by green tea and red wine, by blueberries, blackberries, red grapes, kiwi, watermelon, parsley, and spinach, while two brands of beer, coffee, black tea, rooibos tea, morellos, black-currants, plums, red beets, broccoli (raw and cooked), and chives were somewhat less active. One brand of beer was only moderately active while white wine, bananas, white grapes, and strawberries were inactive. Similarly, genotoxicity of AAF was strongly reduced by green, black, and rooibos tea, red wine, morellos, black-currants, kiwi, watermelon, and spinach while plums, red beets, and broccoli (raw) were less potent. Broccoli cooked exerted only moderate and white wine weak antigenotoxic activity. With respect to the possible mechanism(s) of inhibition of genotoxicity, benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8-OH) and N-OH-PhIP were applied as substrates for the CYP1A family and for rSULT 1C1, respectively. Morellos, black-currants, and black tea strongly reduced the genotoxicity of BaP-7,8-OH, onions, rooibos tea, and red wine were less potent while red beets and spinach were inactive. On the other hand, red beets and spinach strongly inhibited the genotoxicity of N-OH-PhIP, rooibos tea was weakly active while all other items were inactive. These results are suggestive for enzyme inhibition as mechanism of protection by complex mixtures of plant origin. Taken together, our results demonstrate that protection by beverages, fruits, and vegetables against genotoxicity of heterocyclic aromatic amines may take place within metabolically competent mammalian cells as well as under the conditions of the Salmonella/reversion assay.

Heterologous expression of human N-acetyltransferases 1 and 2 and sulfotransferase 1A1 in Salmonella typhimurium for mutagenicity testing of heterocyclic amines.

A variety of carcinogenic heterocylic amines (HAs) are found in cooked food. They can be metabolised to reactive intermediates via N-hydroxylation catalysed by cytochrome P450 1A2, followed by conjugation of the resulting N-hydroxyl group by N-acetyltransferase (NAT) or sulfotransferase (SULT). In order to compare the role of O-acetylation and O-sulfonation by human enzymes in the activation of HAs, we have introduced the cDNAs for wild-type forms of human NAT1, NAT2 and SULT1A1 in the acetyltransferase-deficient Salmonella typhimurium strain TA1538/1,8-DNP. Functional expression of recombinant proteins was demonstrated using immunoblot analysis and determination of enzyme activity with characteristic substrates. The established strains were used to study the mutagenicity of the N-hydroxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The results demonstrate that N-hydroxy-HAs are activated by different human enzymes. At the concentrations used in the mutagenicity assay, N-hydroxy-IQ was activated by human NAT2, but not by NAT1 or SULT1A1. In contrast, N-hydroxy-PhIP was activated specifically by human SULT1A1, but not by NAT1 or NAT2. Therefore, both O-acetylation and O-sulfonation by human enzymes have to be regarded as important determinants for HA genotoxicity in humans.

Structure and localization of the human SULT1B1 gene: neighborhood to SULT1E1 and a SULT1D pseudogene.

The soluble sulfotransferases are involved in the elimination of xenobiotics, the activation of procarcinogens, and the regulation of hormones. They comprise a gene superfamily (SULT). The structure and chromosomal location of nine human SULT genes are known. We have characterized a further gene, SULT1B1. Its structure is similar to that of other SULT1 genes. However, the total length of its eight exons and the introns (33.6 kb) is larger than that of other human SULT1 genes (4 to 21 kb). The SULT1B1 gene sequence is part of a sequence entry in the unfinished High-Throughput Genomic Sequences (HTGS) division of GenBank. However, the order and orientation of the SULT1B1 exons are not correct in this entry. SULT1B1 is located on chromosome 4q13.1, nearly 100 kb downstream of SULT1E1 on the same strand. The intervening sequence contains a SULT-like structure showing substantial homology to the mouse SULT1D1 cDNA recently described. However, in humans this structure represents a pseudogene (SULT1D1P) because of mutated splice donors/acceptors and in-frame stop codons in the sequence corresponding to exon II. This SULT gene cluster is located on the minus strand of chromosome 4 with SULT1B1 being closest to the centromer.

Human cytosolic sulphotransferases: genetics, characteristics, toxicological aspects.

Cytosolic sulphotransferases transfer the sulpho moiety from the cofactor 5'-phosphoadenosine-3'-phosphosulphate (PAPS) to nucleophilic groups of xenobiotics and small endogenous compounds (such as hormones and neurotransmitters). This reaction often leads to products that can be excreted readily. However, other sulpho conjugates are strong electrophiles and may covalently bind with DNA and proteins. All known cytosolic sulphotransferases are members of an enzyme/gene superfamily termed SULT. In humans, 10 SULT genes are known. One of these genes encodes two different enzyme forms due to the use of alternative first exons. Different SULT forms substantially differ in their substrate specificity and tissue distribution. Genetic polymorphisms have been described for three human SULTs. Several allelic variants differ in functional properties, including the activation of promutagens. Only initial results are available from the analysis of SULT allele frequencies in different population groups, e.g. subjects suffering from specific diseases and corresponding controls.

N-Acetyltransferases, sulfotransferases and heterocyclic amine activation in the breast.

Heterocyclic amines are mammary carcinogens in rats and their N-hydroxy metabolites are substrates for subsequent metabolic activation by N-acetyltransferases (NAT) and sulfotransferases (SULT) in man. We investigated the expression of these enzymes in human breast tissue and the relationship between NAT genotype and NAT mRNA expression or enzyme activity. Immunohistochemical staining of sections of breast tissue identified expression of NAT1 and NAT2 protein in human mammary epithelial cells, but not in the stroma. We also measured the formation of DNA adducts of the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in calf thymus DNA after incubation of their promutagenic N-hydroxy metabolites with mammary cytosols prepared from reduction mammoplasty tissue. Experimental observations gained from use of enzyme cofactors and NAT and/or SULT inhibitors on cytosolic enzyme activity, recombinant NAT1 activity and heterocyclic amine-DNA adduct formation suggest that both NAT1 and SULT1A enzymes contribute significantly to the activation of N-hydroxylated heterocyclic amines in mammary tissue. NAT1 mRNA transcript levels were found to be two- to three-fold higher than mRNA transcripts of the NAT2 gene in reduction mammoplasty tissue and mammary epithelial cells. NAT1-specific p-aminobenzoic acid acetylation activity, but not NAT2-specific sulfamethazine acetylation activity, was detectable in mammary cytosols. There was no association apparent between NAT genotype and the levels of NAT mRNA or NAT enzyme activity, or between NAT1 genotype and IQ-DNA adduct formation mediated by mammary cytosols. Western blot analysis of mammary cytosolic protein showed detectable levels of SULT1A1 and SULT1A3.

Proliferation and differentiation of murine haemopoietic progenitor cells in stroma-free culture in the presence of metabolites of chlorinated pesticides.

We have studied the influence of metabolites of chlorinated pesticides (lindane, pentachlorophenol, hexachlorobenzene) on proliferation and differentiation in two stroma-free murine bone marrow culture models, a multipotent progenitor cell line (FDCP-mix) and primary lineage-depleted bone marrow cells. Tetrachlorohydroquinone (Cl(4)pHQ), tetrachloro-p-benzoquinone (Cl(4p)BQ), but not their positional isomers, tetrachlorocatechol (Cl(4)oHQ) and tetrachloro-o-benzoquinone (Cl(4)oBQ), nor 2,4,6-trichlorophenol (2,4,6-Cl(3)P), were much more toxic to FDCP-mix cells cultured under conditions which lead to self-renewal than under conditions which lead to granulocyte-macrophage differentiation. Under the latter conditions, Cl(4)pHQ and Cl(4p)BQ even stimulated growth at intermediate concentration levels. In the primary cell cultures, pronounced differences were observed in the sensitivity between individual developmental pathways and between the different compounds. The percent of cells differentiating into the granulocytic lineage was increased at high concentration levels of each test compound. However, stimulatory effects on the macrophage lineage were observed at intermediate concentration levels of Cl(4)pHQ, Cl(4p)BQ and 2,4,6-Cl(3)P, and differentiation into erythrocytes was stimulated at low concentrations of 2,4,6-Cl(3)P. It is concluded that chlorinated monocyclic pesticides, after biotransformation to quinoid metabolites, may interact directly with haemopoietic progenitor cells with differential effects on self-renewal and differentiation. These mechanisms could lead to myeloplastic disorders.

Heterocyclic amines: human carcinogens in cooked food?

During the frying of meat and fish, genotoxic heterocyclic amines (HCAs) are formed. The dietary exposure to HCAs may be implicated in the aetiology of human cancer, but there may be other factors in our diet that prevent the genotoxic effects of these compounds. Within the project described here, we plan to identify regional and individual cooking habits that affect HCA-levels in our food. These are determined with a validated analytical method and the exposure to HCAs is estimated by dietary assessment. Biomarker analysis will be employed to estimate recent or long-term exposure to HCAs. In order to identify genetically determined risk factors in humans, cell lines are genetically engineered expressing allelic variants of acetyl- and sulfotransferases implicated in HCA metabolism. Species differences of metabolism and toxicity of HCAs are assessed and the influence of the intestinal microflora on HCA-induced toxicity is evaluated. Dietary constituents that may reduce the genotoxicity of HCAs are screened for potential protective effects in in vitro and in vivo model systems. Finally, we will aim at human intervention studies to investigate if these protective factors are relevant for man. The objectives of this project are to estimate and possibly reduce the exposure levels to HCAs in Europe, to identify populations highly susceptible to HCA toxicity, and to reduce the toxic effects of HCAs by protective factors.

Sex differences in the activation of tamoxifen to DNA binding species in rat liver in vivo and in rat hepatocytes in vitro: role of sulfotransferase induction.

Previous work has indicated that metabolic activation of tamoxifen in rat liver cells involves cytochrome P450-mediated alpha-hydroxylation, followed by sulfate ester formation, mediated by hydroxysteroid sulfotransferase a (rHSTa), a member of the SULT2A subfamily, which efficiently metabolizes dehydroepiandrosterone. Because it is known that the expression of rHSTa and other SULT2A forms is substantially higher in female rats than in males, it might be predicted that tamoxifen would be a more potent liver carcinogen in females than in males. Yet tamoxifen has been shown to be equipotent in both sexes. To investigate this paradox, primary cultures of hepatocytes were prepared from Fischer F-344 rats and treated with tamoxifen (10 microM) or alpha-hydroxytamoxifen (1 microM). Rats were also treated with tamoxifen daily by gavage (0.12 mmol/kg/day) for up to 14 days. DNA was isolated from hepatocytes and liver and analyzed by 32P-postlabeling. Liver cytosol fractions were prepared and analyzed for dehydroepiandrosterone sulfotransferase activity and SULT2A protein levels. In tamoxifen-treated hepatocytes and after a single dose of tamoxifen in vivo, DNA adduct formation in male cells was significantly lower than in female cells, 11- and 6-fold, respectively. However, with increasing daily doses of rats with tamoxifen, the adduct level in males increased to a level 89% of that in females by 14 days. Dehydroepiandrosterone sulfotransferase activity in male rat liver cytosols was only 17% of the activity of female cytosols after one dose of tamoxifen but 64% after 14 days of exposure to the compound. This increase in activity correlated with increases in the levels of SULT2A protein, detected by Western blotting. Western blotting did not allow the unambiguous identification of the induced SULT2A form(s). However, by using a specific reverse transcriptase/PCR technique, it was found that it was primarily rHSTa that was induced. Thus, after prolonged exposure to tamoxifen, DNA adduct formation and rHSTa expression in males are significantly closer to the levels in females than they are after initial exposure. These changes explain the similar susceptibility of male and female rats to tamoxifen carcinogenesis.

An overview of bioactivation of chemical carcinogens.

Most environmental carcinogens require metabolic activation to reactive intermediates and are mutagenic in appropriate test systems. During the last decade, the cDNAs of numerous xenobiotic-metabolizing enzymes have been cloned. The individually expressed enzymes were used to study their substrate specificities and their inhibition by other compounds. Various enzymes were expressed directly in target cells of in vitro mutagenicity tests. This is illustrated in the present study for rat and human sulphotransferases (SULTs) expressed in Salmonella typhimurium TA1538. Numerous compounds were mutagenic in the new test system. Some of these promutagens were activated by several different SULT forms, whereas many other promutagens were activated with high selectivity by a specific enzyme form, but not by genetically closely related forms from the same species (e.g. allelic variants) or orthologous enzymes from other species. Similar findings have been made using recombinant test systems for specific forms of other classes of enzymes (e.g. cytochromes P450). This high selectivity in activation (and inactivation) may explain some organotropisms as well as species and inter-individual differences in the action of carcinogens. Many carcinogen-metabolizing enzymes are induced or inhibited by other xenobiotics. Such interactions can be exploited for chemo-prevention, which however may be carcinogen- and tissue-dependent.

Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway explaining the estrogenic activity of PCBs.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants which exert a variety of toxic effects in animals, including disturbances of sexual development and reproductive function. The estrogenic effects of PCBs may be mediated in part by hydroxylated PCB metabolites (PCB-OHs), but the mechanisms by which they are brought about are not understood. PCBs as well as PCB-Hs show low affinities for both alpha and beta estrogen receptor isoforms. In the present study we demonstrate that various environmentally relevant PCB-OHs are extremely potent inhibitors of human estrogen sulfotransferase, strongly suggesting that they indirectly induce estrogenic activity by increasing estradiol bioavailability in target tissues.

Association between functional genetic polymorphisms of human sulfotransferases 1A1 and 1A2.

Three human phenol sulfotransferases, provisionally named SULT1A1, 1A2 and 1A3, show 91-96% homology of their amino acid sequences and are encoded by neighbouring gene loci. Functional genetic polymorphisms are known for two of these sulfotransferases. In SULT1A1, a G to A transition leads to an Arg213 to His exchange and eliminates a Bsp143II restriction site. SULT1A1*His shows lower enzyme activity and thermostability than SULT1A1*Arg. In SULT1A2, an A to C transversion causes an Asn235 to Thr exchange and introduces a BpiI restriction site. Enzyme SULT1A2*Thr is less active than SULT1A2*Asn. These substitutions were detected by restriction fragment length polymorphism analyses of genomic sequences amplified by polymerase chain reaction. Despite the high similarity between the different human SULT1A genes, it was possible to amplify specifically the polymorphic parts of either SULT1A1 or 1A2, but not the homologous sequences of the other SULT, by setting the forward primer into intron 6. DNA from 300 adult male Caucasian subjects was analysed. Allele frequencies were 0.63 and 0.37 for SULT1A1*Arg and *His, and 0.62 and 0.38 for SULT1A2*Asn and *Thr, respectively. The frequency of the haplotype SULT1A1*Arg/SULT1A2*Asn (0.61) was nearly as high as the allele frequencies of its components. The same was observed for the haplotype SULT1A1*His/SULT1A2*Thr, whose frequency was 0.35. In contrast, haplotypes 1A1*Arg/1A2*Thr and 1A1*His/1A2*Asn were very rare. Their frequencies (0.02 each) were less than 10% of the figures expected in an independent distribution. The results demonstrate a strong association of the alleles producing the more active enzyme variants (SULT1A1*Arg and SULT1A2*Asn) and of those encoding the less active variants (SULT1A1*His and SULT1A2*Thr).

Sulfotransferases: genetics and role in toxicology.

The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT). Ten distinct human SULT forms are known. Two SULT forms represent splice variants, the other forms are encoded by separate genes. Common functional polymorphisms of the transcribed region are known for two of the forms. We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays. The expressed SULTs activated numerous compounds to mutagens in both assay systems. However, some promutagens were activated by only one or several of the human SULTs. Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.

Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.

The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen.

Sulfotransferases in the bioactivation of xenobiotics.

Conjugation of xenobiotics is often associated with detoxification. However, this traditional view is one-sided. In particular, numerous compounds are known that are metabolized to chemically reactive metabolites via sulfation (O-sulfonation). This can be rationalized by the fact that the sulfate group is electron-withdrawing and may be cleaved off heterolytically in appropriate molecules, thus leading to the formation of a strongly electrophilic cation. The heterologous expression of sulfotransferases in indicator cells of standard mutagenicity tests has substantially improved the accessibility of this activation pathway. The use of this technology is important, since many reactive sulfate conjugates only show strong toxicological effects if they are generated directly within the indicator cell, due to their insufficient penetration of cell membranes. Xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a superfamily (SULT). Eleven distinct human SULT forms are known, which strongly differ in their tissue distribution and their substrate specificity. Common functionally relevant genetic polymorphisms of the transcribed region are known for two of the forms, SULT1A1 and 1A2. Studies using recombinant test systems demonstrate that many promutagens are activated with high selectivity by an individual SULT form. Pronounced differences in promutagen activation were detected between the different human forms, including their allelic variants, and also between orthologous SULTs from different species. Therefore, SULTs may be involved in the individual genetic disposition, species differences, and organotropisms for toxicological effects of chemicals. Activation by SULTs differs from other activation pathway in its cyclic nature: reaction of a sulfuric acid ester with water usually regenerates the hydroxylated compound, which becomes available for a new cycle of activation. SULT-mediated reactivation may even occur if another initial reactive species, e.g. an epoxide, has reacted with water.