Juices of Brassica vegetables are mutagenic and form characteristic DNA adducts in bacteria and mammalian cells. In this study, we examined whether such adducts are also formed in vivo in animal models. Rats fed raw broccoli ad libitum in addition to normal laboratory chow for 5 weeks showed one major adduct spot and sometimes an additional minor adduct spot in liver, kidney, lung, blood and the gastrointestinal tract, as determined by 32P-postlabelling/thin-layer chromatography. Adducts with the same chromatographic properties were formed when herring sperm DNA (or dG-3’-phosphate) was incubated with 1-methoxy-3-indolylmethyl glucosinolate (phytochemical present in Brassica plants), in the presence of myrosinase (plant enzyme that hydrolyses glucosinolates to bioactive breakdown products). UPLC–MS/MS analysis corroborated this finding: 1-Methoxy-3-indolylmethyl-substituted purine nucleosides were detected in the hepatic DNA of broccoli-fed animals, but not in control animals. Feeding raw cauliflower led to the formation of the same adducts. When steamed rather than raw broccoli was used, the adduct levels were essentially unchanged in liver and jejunum, but elevated in large intestine. Due to inactivation of myrosinase by the steaming, higher levels of the glucosinolates may have reached the large bowl to be activated by glucosidases from intestinal bacteria. In conclusion, the consumption of common Brassica vegetables can lead to the formation of substantial levels of DNA adducts in animal models. The adducts can be attributed to a specific phytochemical, neoglucobrassicin (1-methoxy-3-indolylmethyl glucosinolate).

We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N2-(1-MIM)-dG, N6-(1-MIM)-dA] in six tissues, as well as protein adducts [τN-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC–MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.

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.

Scope: Breakdown products of certain glucosinolates induce detoxifying enzymes and demonstrate preventive activities against chemically induced tumourigenesis in animal models. However, other breakdown products are genotoxic. 1-Methoxy-3-indolylmethyl alcohol (1-MIM-OH) is mutagenic in bacterial and mammalian cells upon activation by sulphotransferases and forms DNA adducts in mouse tissues. This effect is enhanced in mice transgenic for human sulphotransferases 1A1/2 (FVB/N-hSULT1A1/2). Therefore, we explored gene expression changes induced by 1-MIM-OH in mouse liver.Methods and results: FVB/N-hSULT1A1/2 mice were orally treated with 1-MIM-OH for 21 or 90 days, leading to high levels of hepatic 1-MIM-DNA adducts. Genome-wide expression analyses demonstrated no influence on detoxifying enzymes, but up-regulation of many mediators of the tumour suppressor p53 and down-regulation of Fhit and other long genes. While this p53 response might indicate protection, it was unable to prevent the accumulation of DNA adducts. However, various epidemiological studies reported inverse associations between the intake of cruciferous vegetables and cancer. This association may be due to the presence of other glucosinolates with tumour-preventing influences possibly outweighing adverse effects of some metabolites.Conclusion: 1-MIM-OH is a genotoxic substance inducing a gene expression profile similar to the expression signature caused by known genotoxic hepatocarcinogens.

High consumption of Brassica vegetables is considered to prevent especially colon carcinogenesis. The content and pattern of glucosinolates (GSLs) can highly vary among different Brassica vegetables and may, thus, affect the outcome of Brassica intervention studies. Therefore, we aimed to feed mice with diets containing plant materials of the Brassica vegetables broccoli and pak choi. Further enrichment of the diets by adding GSL extracts allowed us to analyze the impact of different amounts (GSL-poor versus GSL-rich) and different patterns (broccoli versus pak choi) of GSLs on inflammation and tumor development in a model of inflammation-triggered colon carcinogenesis (AOM/DSS model). Serum albumin adducts were analyzed to confirm the up-take and bioactivation of GSLs after feeding the Brassica diets for four weeks. In agreement with their high glucoraphanin content, broccoli diets induced the formation of sulforaphane-lysine adducts. Levels of 1-methoxyindolyl-3-methyl-histidine adducts derived from neoglucobrassicin were the highest in the GSL-rich pak choi group. In the colon, the GSL-rich broccoli and the GSL-rich pak choi diet up-regulated the expression of different sets of typical Nrf2 target genes like Nqo1, Gstm1, Srxn1, and GPx2. GSL-rich pak choi induced the AhR target gene Cyp1a1 but did not affect Ugt1a1 expression. Both colitis and tumor number were drastically reduced after feeding the GSL-rich pak choi diet while the other three diets had no effect. GSLs can act anti-inflammatory and anti-carcinogenic but both effects depend on the specific amount and pattern of GSLs within a vegetable. Thus, a high Brassica consumption cannot be generally considered to be cancer-preventive.

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.

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.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate (GLS) occurring in cabbage, broccoli, and other cruciferous plants is a potent mutagen requiring metabolic activation by myrosinase present in plant cells and intestinal bacteria. We previously reported that 1-MIM-GLS and its alcoholic breakdown product 1-MIM-OH, which requires additional activation by sulfotransferases, form DNA adducts in mice. In the present study, the formation of protein adducts was investigated. First, two major adducts obtained after incubation of individual amino acids, serum albumin, or hemoglobin with 1-MIM-GLS in the presence of myrosinase were identified as τN-(1-MIM)-His and πN-(1-MIM)-His using MS and NMR spectroscopy. After the development of a specific detection method using isotope-dilution UPLC-ESI-MS/MS, adduct formation was confirmed in mice after oral treatment with 1-MIM-GLS. Adduct levels were highest in the cecum and colon, somewhat lower in serum albumin and the liver, and also readily detectable in the lung and hemoglobin. On the contrary, oral treatment with 1-MIM-OH produced the highest adduct levels in the liver. The higher ratio of albumin to hemoglobin adducts in 1-MIM-OH- compared to 1-MIM-GLS-treated animals (8.1 versus 3.5) suggests that in 1-MIM-OH-treated animals albumin adducts were produced mostly in the liver, the site of albumin synthesis. The formation of adducts was approximately linear over a range of single oral doses from 20 to 600 μmol/kg body mass. Repeated oral administration of 1-MIM-OH (up to 40 treatments, thrice per week) led to continuous accumulation of hemoglobin adducts, whereas the level of serum albumin adducts remained rather constant, which reflects the different turnover rates of these proteins (t1/2 nearly 1.9 d for serum albumin and 25 d for hemoglobin in the mouse). Accumulation of adducts was also noticed in the lung. Adduct levels were higher, but their accumulation was weaker in the liver and kidney. The method developed will be useful to assess the exposure of humans to reactive metabolites formed from 1-MIM-GLS present in many foods.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary metabolite of Brassicales species, and its breakdown product 1-MIM alcohol are mutagenic in cells in culture after activation by plant β-thioglucosidase and human sulphotransferase, respectively. In the present study, we administered these compounds orally to mice to study time course, dose dependence, tissue distribution and cellular localization of the 1-MIM DNA adducts formed. We used isotope-dilution ultra-performance liquid chromatography–tandem mass spectrometry to quantify the adducts and raised an antiserum for their immunohistochemical localization. Both compounds formed the same adducts, N 2-(1-MIM)-2′-deoxyguanosine and N 6-(1-MIM)-2′-deoxyadenosine, approximately in a 3.3:1 ratio. 1-MIM glucosinolate primarily formed these adducts in the large intestine, with a luminal–basal gradient, probably due to activation by thioglucosidase from intestinal bacteria. 1-MIM alcohol formed higher levels of adduct than the glucosinolate. Unlike after treatment with the glucosinolate, luminal and basal enterocytes were similarly affected in caecum, and liver and stomach were additional important target tissues. Maximal adduct levels were reached 8 h after the administration of both compounds. The hepatic DNA adducts persisted for the entire observation period (48 h), whereas those in large intestine rapidly declined due to cell turnover, as verified by immunohistochemistry. Hepatic adduct formation was focused on the periportal hepatocytes with concomitant depletion of glycogen, p53 activation and p21 induction. Adduct formation in caecum was associated with massive apoptosis, p53 activation and p21 induction, in particular after treatment with 1-MIM alcohol. It remains to be studied whether similar effects occur in humans after the consumption of Brassicales species.

Problemstellung: Im Kontext der humanen nicht-alkoholischen Fettlebererkrankung (NAFLD) gelten entzündliche hepatische Prozesse als mit der Adipositas assoziiert. Eine verknüpfende Rolle spielen dabei von Fettzellen sezernierte Mediatoren, sogenannte Adipozytokine. Dies wurde auch für Chemerin postuliert, ein in jüngerer Zeit identifiziertes Adipozytokin mit chemotaktischer Wirkung auf bestimmte Populationen inflammatorischer Zellen. Erhöhte Serumspiegel von Chemerin in der Vena hepatica leberzirrhotischer Patienten deuten daneben auf eine zusätzliche Expression des Adipozytokins in der Leber hin. Dies wurde für den Menschen bisher allerdings nicht belegt. Gegenstand dieser Studie war daher die Untersuchung der hepatischen Genexpression von Chemerin und dessen Rezeptor CMKLR1 bei Patienten mit histopathologisch nachgewiesener NAFLD im Vergleich zu Kontrollpatienten ohne NAFLD.

OBJECTIVE:Adipose tissue-derived factors link non-alcoholic fatty liver disease (NAFLD) with obesity, which has also been reported for circulating chemerin. On the other hand, hepatic chemerin and chemokine-like receptor 1 (CMKLR1) mRNA expression has not yet been studied in an extensively characterized patient collective. DESIGN:This study was cross-sectional and experimental in design. METHODS:Liver tissue samples were harvested from 47 subjects and histologically examined according to the NAFLD activity score (NAS). The concentrations of chemerin and CMKLR1 were measured using semi-quantitative real-time PCR, and the concentration of serum chemerin was measured using ELISA. To evaluate potential effects of chemerin and CMKLR1, cultured primary human hepatocytes (PHHs) were exposed to selected metabolites known to play a role in NAFLD (insulin, glucagon, palmitoic acid, and interleukin-6 (IL6)). RESULTS:Chemerin and CMKLR1 mRNA levels were elevated in the human liver. Their expression was correlated with the NAS (R(2)=0.543; P<0.001 and R(2)=0.355; P=0.014 respectively) and was significantly elevated in patients with definite non-alcoholic steatohepatitis (NASH) (P<0.05 respectively). Linear regression analysis confirmed an independent association of liver fibrosis, steatosis, inflammation, and hepatocyte ballooning with hepatic chemerin mRNA expression (P<0.05 respectively). The expression of hepatic chemerin and CMKLR1 was correlated with the measures of obesity (P<0.05). The incubation of PHHs with IL6 significantly increased the expression of CMKLR1 mRNA (P=0.027), while that of chemerin remained unaffected (P>0.05). None of the other metabolites showed an influence (P>0.05). CONCLUSION:This is the first study to show that chemerin mRNA expression is significantly elevated in the liver of NASH patients and that CMKLR1 expression is upregulated in liver inflammation, whereby IL6 could play a causal role.

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (-Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the -Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to -Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.

Objective: Inflammatory states of non-alcoholic fatty liver disease (NAFLD) are associated with obesity via adipocytokines. Circulating levels of the adipocytokine chemerin are in vivo associated with NAFLD and hepatic cirrhosis. However, hepatic gene transcription of chemerin and its receptor CMKLR1 are rarely studied, especially in human NAFLD.

Fragestellung: Die AMPK, ein ubiquitärer zellulärer Energiesensor und das Ziel der Metformintherapie, spielt im Leberstoffwechsel eine zentrale Rolle und reguliert u.a. die Glukoseproduktion und die de novo Lipogenese. In Tiermodellen ist für NAFLD und Diabetes mellitus Typ 2 (DMT2) eine reduzierte Phosphorylierung an Thr172 der katalytischen α Untereinheit der AMPK und eine daraus resultierende Aktivitätsminderung ohne Veränderung der Gesamtproteinmenge gezeigt. Adiponektin gilt als Aktivator der AMPK und vermittelt seine Wirkung zellulär durch die Rezeptoren AdipoR1 und R2. Der Zusammenhang zwischen AMPK und Adiponektin in der humanen NAFLD±DMT2 stand im Fokus dieser Untersuchung.

SCOPE It was reported that the Maillard product 5-hydroxymethylfurfural (HMF) initiates and promotes aberrant crypt foci (ACF) in rat colon. We studied whether 5-sulfooxymethylfurfural (SMF), an electrophilic and mutagenic metabolite of HMF, is able to induce ACF in two murine models. METHODS AND RESULTS In the first model, FVB/N mice received four intraperitoneal administrations of SMF (62.5 or 125 mg/kg) or azoxymethane (10 mg/kg). Animals were killed 4-40 weeks after the last treatment. A total of 1064 ACF and five adenocarcinomas were detected in the azoxymethane-treated groups (20 animals), but none in the negative control and SMF-treated groups (35 and 50 animals, respectively). In the second model, HMF was administered via drinking water to wild-type FVB/N mice and transgenic mice carrying several copies of human sulfotransferase (SULT) 1A1 and 1A2 genes. HMF SULT activity was clearly elevated in cytosolic fractions of colon mucosa, liver and kidney of transgenic animals compared to wild-type mice and humans. The animals (six per group) received 134 and 536 mg HMF/kg/day for 12 weeks. HMF did not induce any ACF either in wild-type or transgenic animals. CONCLUSION We found no evidence for an induction of ACF by HMF or its metabolite SMF in extensive studies in mice.

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, present at substantial levels in several food crops (e.g., broccoli and cabbage), forms DNA adducts in vitro and is mutagenic to bacterial and mammalian cells after activation by the plant enzyme myrosinase. Moreover, a breakdown product, 1-MIM alcohol, is metabolized to a secondary reactive intermediate by some mammalian sulfotransferases (SULTs). First, we incubated herring-sperm DNA with 1-MIM glucosinolate in the presence of myrosinase. We identified and synthesized the predominant adducts, N2-(1-MIM)-dG and N6-(1-MIM)-dA, and developed an UPLC-ESI-MS/MS method for their specific detection using isotopic dilution. Second, we demonstrated both DNA adducts in target cells (Salmonella typhimurium TA100 and Chinese hamster V79) of standard mutagenicity tests treated with 1-MIM glucosinolate/myrosinase as well as in 1-MIM alcohol-treated Salmonella and V79 cells engineered for expression of human SULT1A1. Similar excesses of N2-(1-MIM)-dG over N6-(1-MIM)-dA adducts were found in all cellular models independent of the test compound (1-MIM glucosinolate or alcohol), whereas dA adducts predominated in the cell-free system. Finally, we detected both DNA adducts in colon tissue of a mouse orally treated with 1-MIM glucosinolate. We are going to use this specific and sensitive method for investigating genotoxic risks of food-borne exposure to 1-MIM glucosinolate in animal and human studies.

Mass spectrometric analyses of DNA adducts usually require enzymatic digestion of the DNA to nucleosides. The digestive enzymes used in our laboratory included a calf spleen phosphodiesterase, whose marketing was stopped recently. Using DNA adducted with bioactivated methyleugenol and 1-methoxy-3-indolylmethyl glucosinolate-each forming dA and dG adducts-we demonstrate that replacement of calf spleen phosphodiesterase (Merck) with bovine spleen phosphodiesterase (Sigma-Aldrich) leads to unchanged results. Enzyme levels used for DNA digestion are extremely variable in different studies. Therefore, we sequentially varied the level of each of the three enzymes used. All dose (enzyme)-response (adduct level) curves involved a long plateau starting below the enzyme levels employed previously. Thus, we could reduce the amounts of micrococcal nuclease, phosphodiesterase, and alkaline phosphatase for quantitative DNA digestion by factors of 4, 2, and 333, respectively, compared to our previous protocols. Moreover, we observed significant phosphatase activity of both phosphodiesterase preparations used, which may affect the recovery of adducts with methods requiring digestion to 2'-deoxynucleoside-3'-monophosphates (e.g., (32)P-postlabeling). In addition, the phosphodiesterase from Sigma-Aldrich, but not that from Merck, deaminated dA. This was irrelevant for the dA adducts studied, involving bonding at N(6), but might complicate the analysis of other dA adducts.

Friedreich's ataxia is an inherited neurodegenerative disease caused by the reduced expression of the mitochondrially active protein frataxin. We have previously shown that mice with a hepatocyte-specific frataxin knockout (AlbFxn(-/-)) develop multiple hepatic tumors in later life. In the present study, hepatic carbohydrate metabolism in AlbFxn(-/-) mice at an early and late life stage was analyzed. In young (5-week-old) AlbFxn(-/-) mice hepatic ATP, glucose-6-phosphate and glycogen levels were found to be reduced by ∼74, 80 and 88%, respectively, when compared with control animals. This pronounced ATP, G6P and glycogen depletion in the livers of young mice reverted in older animals: while half of the mice die before 30 weeks of age, the other half reaches 17 months of age and exhibits glycogen, G6P and ATP levels similar to those in age-matched controls. A key event in this respect seems to be the up-regulation of GLUT1, the predominant glucose transporter in fetal liver parenchyma, which became evident in AlbFxn(-/-) mice being 5-12 weeks of age. The most significant histological findings in animals being 17 or 22 months of age were the appearance of multiple clear cell, mixed cell and basophilic foci throughout the liver parenchyma as well as the development of hepatocellular adenomas and carcinomas. The hepatocarcinogenic process in AlbFxn(-/-) mice shows remarkable differences regarding carbohydrate metabolism alterations when compared with all other chemically and virally driven liver cancer models described up to now.