Formation of DNA Adducts by 1-Methoxy-3-indolylmethylalcohol, a Breakdown Product of a Glucosinolate, in the Mouse: Impact of the SULT1A1 Status-Wild-Type, Knockout or Humanised.

We previously found that feeding rats with broccoli or cauliflower leads to the formation of characteristic DNA adducts in the liver, intestine and various other tissues. We identified the critical substances in the plants as 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate and its degradation product 1-MIM-OH. DNA adduct formation and the mutagenicity of 1-MIM-OH in cell models were drastically enhanced when human sulfotransferase (SULT) 1A1 was expressed. The aim of this study was to clarify the role of SULT1A1 in DNA adduct formation by 1-MIM-OH in mouse tissues in vivo. Furthermore, we compared the endogenous mouse Sult1a1 and transgenic human SULT1A1 in the activation of 1-MIM-OH using genetically modified mouse strains. We orally treated male wild-type (wt) and Sult1a1-knockout (ko) mice, as well as corresponding lines carrying the human SULT1A1-SULT1A2 gene cluster (tg and ko-tg), with 1-MIM-OH. N2-(1-MIM)-dG and N6-(1-MIM)-dA adducts in DNA were analysed using isotope-dilution UPLC-MS/MS. In the liver, caecum and colon adducts were abundant in mice expressing mouse and/or human SULT1A1, but were drastically reduced in ko mice (1.2-10.6% of wt). In the kidney and small intestine, adduct levels were high in mice carrying human SULT1A1-SULT1A2 genes, but low in wt and ko mice (1.8-6.3% of tg-ko). In bone marrow, adduct levels were very low, independently of the SULT1A1 status. In the stomach, they were high in all four lines. Thus, adduct formation was primarily controlled by SULT1A1 in five out of seven tissues studied, with a strong impact of differences in the tissue distribution of mouse and human SULT1A1. The behaviour of 1-MIM-OH in these models (levels and tissue distribution of DNA adducts; impact of SULTs) was similar to that of methyleugenol, classified as "probably carcinogenic to humans". Thus, there is a need to test 1-MIM-OH for carcinogenicity in animal models and to study its adduct formation in humans consuming brassicaceous foodstuff.

In Silico Prediction of Human Sulfotransferase 1E1 Activity Guided by Pharmacophores from Molecular Dynamics Simulations.

Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.

Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels.

Methyleugenol is a rodent hepatocarcinogen occurring in many herbs and spices as well as essential oils used for flavoring. Following metabolic activation by cytochromes P450 (CYPs) and sulfotransferases (SULTs), methyleugenol can form DNA adducts. Previously, we showed that DNA adduct formation by methyleugenol in mouse liver is dependent on SULT1A1 expression and that methyleugenol DNA adducts are abundant in human liver specimens. In humans, SULT1A1 activity is affected by genetic polymorphisms, including single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). Here we investigated the relationship between individual methyleugenol DNA adduct levels and SULT1A1 in human liver samples. Using isotope-dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry, we quantified methyleugenol DNA adducts in 121 human surgical liver samples. Frequent CNVs, including deletions (f = 3.3%) and duplications (f = 36.4%) of SULT1A1, were identified using qPCR and TaqMan assays in the donors' genomic DNA. SULT1A1 mRNA and protein levels were quantified using microarray data and Western blot analysis, respectively. Methyleugenol DNA adducts were detected in all 121 liver samples studied. Their levels varied 122-fold between individuals and were significantly correlated to both mRNA and protein levels of SULT1A1 (r s = 0.43, and r s = 0.44, respectively). Univariate and multivariate statistical analysis identified significant associations of SULT1A1 CNVs with mRNA (p = 1.7 × 10-06) and protein (p = 4.4 × 10- 10) levels as well as methyleugenol DNA adduct levels (p = 0.003). These data establish the importance of SULT1A1 genotype for hepatic methyleugenol DNA adducts in humans, and they confirm a strong impact of SULT1A1 CNVs on SULT1A1 hepatic phenotype.

Impact of genetic modulation of SULT1A enzymes on DNA adduct formation by aristolochic acids and 3-nitrobenzanthrone.

Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1-SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(-/-) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(-/-) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(-/-) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation.

Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans.

Bioactivation of food genotoxicants 5-hydroxymethylfurfural and furfuryl alcohol by sulfotransferases from human, mouse and rat: a comparative study.

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are moderately potent rodent carcinogens that are present in thermally processed foodstuffs. The carcinogenic effects were hypothesized to originate from sulfotransferase (SULT)-mediated bioactivation yielding DNA-reactive and mutagenic sulfate esters, a confirmed metabolic pathway of HMF and FFA in mice. It is known that orthologous SULT forms substantially differ in substrate specificity and tissue distribution. This could influence HMF- and FFA-induced carcinogenic effects. Here, we studied HMF and FFA sulfoconjugation by 30 individual SULT forms of humans, mice and rats. The catalytic efficiencies (k cat/K M) of HMF sulfoconjugation of human SULT1A1 (13.7 s(-1) M(-1)), mouse Sult1a1 (15.8 s(-1) M(-1)) and 1d1 (4.8 s(-1) M(-1)) and rat Sult1a1 (5.3 s(-1) M(-1)) were considerably higher than those of all other SULT forms investigated (≤0.73 s(-1 )M(-1)). FFA sulfoconjugation was monitored using adenosine as a nucleophilic scavenger for the reactive 2-sulfoxymethylfuran (t 1/2 = 20 s at 37 °C). The resulting adduct N (6)-((furan-2-yl)methyl)-adenosine (N (6)-MF-A) was quantified by isotope-dilution UPLC-MS/MS. The rates of N (6)-MF-A formation showed that hSULT1A1 and its orthologues in mice and rats were also the most important contributors to FFA sulfoconjugation in each of the species. Taken together, the catalytic capacity of hSULT1A1 is comparable to that of mSult1a1 in mice, the species in which carcinogenic effects of HMF and FFA were detected. This is of primary concern due to the expression of hSULT1A1 in many different tissues.

The effect of knockout of sulfotransferases 1a1 and 1d1 and of transgenic human sulfotransferases 1A1/1A2 on the formation of DNA adducts from furfuryl alcohol in mouse models.

Furfuryl alcohol is a rodent carcinogen present in numerous foodstuffs. Sulfotransferases (SULTs) convert furfuryl alcohol into the DNA reactive and mutagenic 2-sulfoxymethylfuran. Sensitive techniques for the isotope-dilution ultra performance liquid chromatography-tandem mass spectrometry quantification of resulting DNA adducts, e.g. N (2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N (2)-MF-dG), were developed. To better understand the contribution of specific SULT forms to the genotoxicity of furfuryl alcohol in vivo, we studied the tissue distribution of N (2)-MF-dG in different mouse models. Earlier mutagenicity studies with Salmonella typhimurium strains expressing different human and murine SULT forms indicated that human SULT1A1 and murine Sult1a1 and 1d1 catalyze furfuryl alcohol sulfo conjugation most effectively. Here, we used three mouse lines to study the bioactivation of furfuryl alcohol by murine SULTs, FVB/N wild-type (wt) mice and two genetically modified models lacking either murine Sult1a1 or Sult1d1. The animals received a single dose of furfuryl alcohol, and the levels of the DNA adducts were determined in liver, kidney, lung, colon and small intestine. The effect of Sult1d1 gene disruption on the genotoxicity of furfuryl alcohol was moderate and limited to kidney and small intestine. In contrast, the absence of functional Sult1a1 had a massive influence on the adduct levels, which were lowered by 33-73% in all tissues of the female Sult1a1 null mice compared with the wt animals. The detection of high N (2)-MF-dG levels in a humanized mouse line expressing hSULT1A1/1A2 instead of endogeneous Sult1a1 and Sult1d1 supports the hypothesis that furfuryl alcohol is converted to the mutagenic 2-sulfoxymethylfuran also in humans.

Formation of hepatic DNA adducts by methyleugenol in mouse models: drastic decrease by Sult1a1 knockout and strong increase by transgenic human SULT1A1/2.

Methyleugenol--a natural constituent of herbs and spices--is hepatocarcinogenic in rodent models. It can form DNA adducts after side-chain hydroxylation and sulfation. We previously demonstrated that human sulfotransferases (SULTs) 1A1 and 1A2 as well as mouse Sult1a1, expressed in Salmonella target strains, are able to activate 1'-hydroxymethyleugenol (1'-OH-ME) and 3'-hydroxymethylisoeugenol (3'-OH-MIE) to mutagens. Now we investigated the role of these enzymes in the formation of hepatic DNA adducts by methyleugenol in the mouse in vivo. We used FVB/N mice [wild-type (wt)] and genetically modified strains in this background: Sult1a1 knockout (ko), transgenic for human SULT1A1/2 (tg) and the combination of both modifications (ko-tg). Methyleugenol (50mg/kg body mass) formed 23, 735, 3770 and 4500 N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adducts per 10(8) 2'-deoxyribonucleosides (dN) in ko, wt, ko-tg and tg mice, respectively. The corresponding values for an equimolar dose of 1'-OH-ME were 12, 1490, 12 400 and 13 300 per 10(8) dN. Similar relative levels were observed for the minor adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Thus, the adduct formation by both compounds was nearly completely dependent on the presence of SULT1A enzymes, with human SULT1A1/2 producing stronger effects than mouse Sult1a1. Moreover, a dose of 0.05 mg/kg methyleugenol (one-fourth of the estimated average daily exposure of humans) was sufficient to form detectable adducts in humanized (ko-tg) mice. Although 3'-OH-MIE was equally mutagenic to 1'-OH-ME in Salmonella strains expressing human SULT1A1 or 1A2, it only formed 0.14% of hepatic adducts in ko-tg mice compared with an equimolar dose of 1'-OH-ME, suggesting an important role of detoxifying pathways for this isomer in vivo.

Determination of sulfotransferase forms involved in the metabolic activation of the genotoxicant 1-hydroxymethylpyrene using bacterially expressed enzymes and genetically modified mouse models.

1-Methylpyrene, a carcinogenic polycyclic aromatic hydrocarbon, forms benzylic DNA adducts, in particular N2-(1-methylpyrenyl)-2'-deoxyguanosine, in mice and rats. It is bioactivated via 1-hydroxymethylpyrene (1-HMP) to electrophilic 1-sulfooxymethylpyrene (1-SMP). In this study, we explored the role of individual mouse sulfotransferase (SULT) forms in this activation. First, we showed that all nine mouse SULTs tested were able to activate 1-HMP to a mutagen in the his- Salmonella typhimurium reversion test. Some activation was even observed with Sult2a3 and Sult5a1, orphan forms for which no substrates were identified hitherto. Subsequently, we used cytosolic preparations from tissues of four mouse lines (wild-type, Sult1a1-, Sult1d1-, and transgenic for human SULT1A1/2) for the activation of 1-HMP in the mutagenicity assay. The most prominent impacts of the genetic SULT status were 96% decrease in hepatic activation by Sult1a1 knockout, 99% decrease in renal activation by Sult1d1 knockout, and 100-fold increase in pulmonary activation by transgenic human SULT1A1/2. Finally, we treated the various mouse lines with 1-HMP (19.3 mg/kg, intraperitoneally), and then determined 1-SMP levels in plasma and DNA adducts in tissues. Transgenic human SULT1A1/2 strongly enhanced 1-SMP plasma levels and DNA adduct formation in the liver, lung, heart, and kidney but not in the colon. Sult1a1 and Sult1d1 knockout reduced plasma 1-SMP levels as well as DNA adduct formation in some tissues (strongest effects: 97% decrease in 1-SMP and 89% decrease in hepatic adducts in Sult1a1- mice). The adduct levels detected in various tissues did not accurately reflect the activation capacity of these tissues determined in vitro, probably due to the distribution of the reactive metabolite 1-SMP via the circulation. In conclusion, we demonstrated that many mouse SULT forms are able to activate 1-HMP. In vivo, we verified a prominent role of Sult1a1 in hepatic and renal adduct formation and a smaller but unambiguous role of Sult1d1, and demonstrated the strong impact of transgenic human SULT1A1/2.

The carcinogen 1-methylpyrene forms benzylic DNA adducts in mouse and rat tissues in vivo via a reactive sulphuric acid ester.

The common polycyclic aromatic hydrocarbon 1-methylpyrene is hepatocarcinogenic in the newborn mouse assay. In vitro studies showed that it is metabolically activated via benzylic hydroxylation and sulphation to a reactive ester, which forms benzylic DNA adducts, N(2)-(1-methylpyrenyl)-2'-deoxyguanosine (MPdG) and N(6)-(1-methylpyrenyl)-2'-deoxyadenosine (MPdA). Formation of these adducts was also observed in animals treated with the metabolites, 1-hydroxymethylpyrene and 1-sulphooxymethylpyrene (1-SMP), whereas corresponding data are missing for 1-methylpyrene. In the present study, we treated mice with 1-methylpyrene and subsequently analysed blood serum for the presence of the reactive metabolite 1-SMP and tissue DNA for the presence of MPdG and MPdA adducts. We used wild-type mice and a mouse line transgenic for human sulphotransferases (SULT) 1A1 and 1A2, males and females. All analyses were conducted using ultra-performance liquid chromatography coupled with tandem mass spectrometry, for the adducts with isotope-labelled internal standards. 1-SMP was detected in all treated animals. Its serum level was higher in transgenic mice than in the wild-type (p < 0.001). Likewise, both adducts were detected in liver, kidney and lung DNA of all exposed animals. The transgene significantly enhanced the level of each adduct in each tissue of both sexes (p < 0.01-0.001). Adduct levels were highest in the liver, the target tissue of carcinogenesis, in each animal model used. MPdG and MPdA adducts were also observed in rats treated with 1-methylpyrene. Our findings corroborate the hypothesis that 1-SMP is indeed the ultimate carcinogen of 1-methylpyrene and that human SULT are able to mediate the terminal activation in vivo.

Sulfotransferase-independent genotoxicity of illudin S and its acylfulvene derivatives in bacterial and mammalian cells.

Acylfulvenes are a class of antitumor agents derived from illudin S, a sesquiterpenoid toxin isolated from mushrooms of the genus Omphalotus. Although DNA appears to be their major target, no data concerning mutagenicity of acylfulvenes are available in the literature, and limited data have been published on illudin S. Enzyme-mediated biotransformations have been demonstrated to influence the cytotoxicity of acylfulvenes. Illudin S and some acylfulvenes [e.g., (-)-6-hydroxymethylacylfulvene (HMAF)] are allylic alcohols with potential for enhanced cytotoxicity and genotoxicity by means of metabolic sulfation. Therefore, we studied the influence of various heterologously expressed human sulfotransferases (SULTs) on biological activities of illudin S and HMAF in bacterial and mammalian cells. (-)-Acylfulvene (AF) was tested as a congener lacking an allylic hydroxyl group. We found: (1) all three compounds were mutagenic in standard Salmonella typhimurium strains TA98, TA100 and TA104; (2) they induced gene mutations (at the hypoxanthine phosphoribosyl transferase locus) and sister chromatid exchange (SCE) in Chinese hamster V79 cells; (3) these effects were practically unaffected when human SULTs were expressed in the target bacteria or mammalian cells (using SCE as the endpoint); (4) illudin S demonstrated 40-600 times higher genotoxic activities than the semisynthetic acylfulvenes studied; it was positive in the SCE test even at a concentration of 0.3 nM; (5) genotoxicity in mammalian cells was observed at substantially lower concentrations of the compounds than required for a positive result in the bacterial test (400 nM with illudin S). We conclude that illudin S, HMAF and AF are potent genotoxicants and human SULTs do not play a significant role in their bioactivation.

Highly selective bioactivation of 1- and 2-hydroxy-3-methylcholanthrene to mutagens by individual human and other mammalian sulphotransferases expressed in Salmonella typhimurium.

The benzylic alcohols 1- and 2-hydroxy-3-methylcholanthrene (OH-MC) are major primary metabolites of the carcinogen 3-methylcholanthrene (MC). We investigated them for mutagenicity in TA1538-derived Salmonella typhimurium strains expressing mammalian sulphotransferases (SULTs). 1-OH-MC was efficiently activated by human (h) SULT1B1 (2400 revertants/nmol), weakly activated by hSULT1C3 and hSULT2A1 (2-9 revertants/nmol), but not activated by the other hSULTs studied (1A2, 1A3, 1C2 and 1E1). Mouse, rat and dog SULT1B1 activated 1-OH-MC (8-100 revertants/nmol) with much lower efficiency than their human orthologue. The other isomer, 2-OH-MC, was activated to a potent mutagen by hSULT1A1 (4000-5400 revertants/nmol), weakly activated by hSULT1A2 or hSULT2A1 (1-12 revertants/nmol), but not activated by the other hSULTs. In contrast to their human orthologue, mouse, rat and dog SULT1A1 did not appreciably activate 2-OH-MC (<1 to 6 revertants/nmol), either. Instead, mouse and rat SULT1B1, unlike their human and canine orthologues, demonstrated some activation of 2-OH-MC (15-100 revertants/nmol). Docking analyses indicated that 1- and 2-OH-MC might bind to the active site of hSULT1A1 and hSULT1B1, but only for (S)-2-OH-MC/hSULT1A1 and (R)-1-OH-MC/hSULT1B1 with an orientation suitable for catalysis. Indeed, 1- and 2-OH-MC were potent inhibitors of the hSULT1A1-mediated sulphation of acetaminophen [concentration inhibiting the enzyme activity by 50% (IC50) 15 and 13nM, respectively]. This inhibition was weak with mouse, rat and dog SULT1A1 (IC50 ≥ 4 µM). Inhibition of the SULT1B1 enzymes was moderate, strongest for 1-OH-MC/hSULT1B1. In conclusion, this study provides examples for high selectivity of bioactivation of promutagens by an individual form of human SULT and for pronounced differences in activation capacity between orthologous SULTs from different mammalian species. These characteristics make the detection and evaluation of such mutagens extremely difficult, in particular as the critical form may even differ for positional isomers, such as 1- and 2-OH-MC. Moreover, the species-dependent differences will complicate the verification of in vitro results in animal studies.

Intestinal carcinogenesis of two food processing contaminants, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 5-hydroxymethylfurfural, in transgenic FVB min mice expressing human sulfotransferases.

Humans express sulfotransferases (SULTs) of the SULT1A subfamily in many tissues, whilst the single SULT1A gene present in rodents is mainly expressed in liver. The food processing contaminants, 5-hydroxymethylfurfural (HMF) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are bioactivated by human SULT1A1 and SULT1A2. FVB multiple intestinal neoplasia (Min) mice, which spontaneously develop tumors and flat aberrant crypt foci (ACF) in intestine, were crossed with transgenic FVB mice expressing human SULT1A1 and 1A2 (hSULT) in several tissues, giving rise to wild-type and Min mice with and without hSULT. One-week-old Min mice with or without hSULT were given HMF (375 or 750 mg/kg bw) or saline by gavage three times a week for 11 wk. In another experiment, the F1 generation received subcutaneous injections of 50 mg/kg bw PhIP or saline 1 wk before birth, and 1, 2, and 3 wk after birth. HMF did not affect the formation of tumors, but may have induced some flat ACF (incidence 15-20%) in Min mice with and without hSULT. No control mouse developed any flat ACF. With the limitation that these putative effects were weak, they were unaffected by hSULT expression. The carcinogenic effect of PhIP increased in the presence of hSULT, with a significant increase in both incidence (31-80%) and number of colonic tumors (0.4-1.3 per animal). Thus, intestinal expression of human SULT1A1 and 1A2 might increase the susceptibility to compounds bioactivated via this pathway implying that humans might be more susceptible than conventional rodent models.

Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium strains engineered for expression of various human and rodent sulphotransferases.

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are present in numerous foodstuffs at high levels. FFA is also used for the production of polymers. Both compounds had demonstrated some evidence of carcinogenic activity in 2-year bioassays. We tested these compounds and four congeners for mutagenicity in Salmonella typhimurium TA100 and TA100-derived strains expressing human or rodent sulphotransferases (SULTs). 5-Hydroxymethylfuroic acid, a metabolite of HMF, was not mutagenic in any strain. 3-Hydroxymethylfuran was weakly mutagenic in all strains independently of SULT expression. HMF, 2,5-(bishydroxymethyl)furan (metabolite of HMF), FFA and 5-methyl-FFA were inactive in TA100 but strongly mutagenic when human SULT1C2 was expressed. This form has been detected in ovary, kidney and foetal tissues. Human SULT1A1, SULT1A2 and SULT1A3 as well as murine Sult1a1 and Sult1d1 also activated some hydroxymethyl-substituted furans to varying degrees. Whereas chemically synthesised 5-sulphooxymethylfurfural was mutagenic in TA100, furfuryl sulphate was bacteriotoxic, only leading to marginal increases in the number of revertants. Furfuryl acetate, an uncharged ester of FFA, used as fragrance and food flavouring, was clearly mutagenic. We determined half-life times of 120 min, 20 s and 10 h, respectively, for 5-sulphooxymethylfurfural, furfuryl sulphate and furfuryl acetate at 37°C in water. It is likely that the short lifespan of furfuryl sulphate, together with its charge, led to insufficient penetration of the bacteria when added externally, although it was mutagenic when generated by appropriate SULTs from FFA within the cell.

Altered tissue distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts in mice transgenic for human sulfotransferases 1A1 and 1A2.

Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP-DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP.

Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides.

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

Constitutive expression of bioactivating enzymes in normal human prostate suggests a capability to activate pro-carcinogens to DNA-damaging metabolites.

BACKGROUND: The constitutive bioactivating capacity of human prostate may play a role in determining risk of adenocarcinoma developing in this tissue. Expression of candidate enzymes that convert exogenous and/or endogenous agents into reactive DNA-damaging species would suggest the potential to generate initiating events in prostate cancer (CaP). METHODS: Normal prostate tissues from UK-resident Caucasians (n = 10) were collected following either radical retropubic prostatectomy (RRP) or cystaprostatectomy (CyP). An analysis of gene and protein expression of candidate metabolizing enzymes, including cytochrome P450 (CYP)1A1, CYP1A2, CYP1B1, N-acetyltransferase 1 (NAT1), sulfotransferase (SULT)1A1, SULT1A3, NAD(P)H:quinone oxidoreductase (NQO1), prostaglandin H synthase 1 (cyclooxygenase 1; COX1), and CYP oxidoreductase (POR) was carried out. Quantitative real-time reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemical analysis were conducted. RESULTS: Except for CYP1A1 and CYP1A2, the metabolizing enzymes examined appeared to be expressed with minimal inter-individual variation (in general, approximately two- to fivefold) in the expression levels. Enzymes such as CYP1B1 and NQO1 that are capable of bioactivating pro-carcinogens to reactive metabolites were readily identifiable in human prostate. Immunohistochemical analysis showed that although some expression is located in the stroma, the majority is localized to epithelial cells lining the glandular elements of the tissue; these are the cells from which CaP might arise. CONCLUSION: Constitutive expression of bioactivating enzymes confers the potential to convert a range of exogenous and/or endogenous agents to reactive species capable of inducing DNA damaging events. These findings suggest an organ capability for pro-carcinogen activation that could play an important role in the etiology of human CaP.

Phase II metabolism of hesperetin by individual UDP-glucuronosyltransferases and sulfotransferases and rat and human tissue samples.

Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3' and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3'-O-glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3'-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3'-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo.

Impact of gut microbiota on intestinal and hepatic levels of phase 2 xenobiotic-metabolizing enzymes in the rat.

Using immunoblotting, we compared levels of phase 2 enzymes in liver, small intestine, cecum, and colon of germ-free and control rats (reassociated with rat intestinal microbiota). In addition, colonic levels were studied after association with human intestinal microbiota. The glutathione transferases (GSTs) studied, gastrointestinal glutathione peroxidase (GPX2), both epoxide hydrolases (EPHXs), and N-acetyltransferase (NAT) 1, were detected in all tissues. GPX2 and GSTP1 were highest in large bowel; the other enzymes of this group were highest in liver. NAT2 was found in the large bowel but not in the liver or small bowel. Sulfotransferases (SULTs) were detected in liver but were absent in small intestine; two forms were present at moderate levels in the large intestine. Strong gender-dependent differences were observed for several enzymes in liver but not in gut. Colonic levels in germ-free animals differed from those in control animals (* indicates statistical significance) for GSTA1/2 (4.0*- and 5.0*-fold in males and females, respectively), GSTA4 (1.5*/1.9*-fold), GSTM1 (1.1/1.5*-fold), EPHX1 (3.5*/2.4*-fold), EPHX2 (1.4/2.1*-fold), SULT1B1 (0.4*/0.6*-fold), SULT1C2 (1.3/1.6*-fold), and NAT2 (1.4/1.5*-fold). Smaller effects were observed when rats were colonized with human, compared with rat, intestinal bacteria. Cecal enzyme levels in germ-free rats were changed similarly to those in colon. No effects were seen in small intestine. In liver, SULT1A1, SULT1C1, and SULT1C2 were elevated in germ-free animals of both genders (1.5- to 2.6-fold); hepatic EPHX2 was elevated 1.6-fold in females. In conclusion, intestinal microbiota can affect levels of xenobiotic-metabolizing enzymes in large intestine and liver, but the effects observed were moderate compared with tissue-dependent expression differences.

Non-enzymatic cleavage of Hsp90 by oxidative stress leads to actin aggregate formation: A novel gain-of-function mechanism.

Aging is accompanied by the accumulation of oxidized proteins. To remove them, cells employ the proteasomal and autophagy-lysosomal systems; however, if the clearance rate is inferior to its formation, protein aggregates form as a hallmark of proteostasis loss. In cells, during stress conditions, actin aggregates accumulate leading to impaired proliferation and reduced proteasomal activity, as observed in cellular senescence. The heat shock protein 90 (Hsp90) is a molecular chaperone that binds and protects the proteasome from oxidative inactivation. We hypothesized that in oxidative stress conditions a malfunction of Hsp90 occurs resulting in the aforementioned protein aggregates. Here, we demonstrate that upon oxidative stress Hsp90 loses its function in a highly specific non-enzymatic iron-catalyzed oxidation event and its breakdown product, a cleaved form of Hsp90 (Hsp90cl), acquires a new function in mediating the accumulation of actin aggregates. Moreover, the prevention of Hsp90 cleavage reduces oxidized actin accumulation, whereas transfection of the cleaved form of Hsp90 leads to an enhanced accumulation of oxidized actin. This indicates a clear role of the Hsp90cl in the aggregation of oxidized proteins.