Nutrition in adult and childhood cancer: role of carcinogens and anti-carcinogens.

There is no doubt that diet is one of the main modifiable risk factors for many degenerative diseases, including cancer. More than 30% of adult cancers can be prevented or delayed by diet, being physically active and having a healthy body weight. Plant-based foods, including fruit, vegetables, and whole grains, a favorable omega-6/omega-3 polyunsaturated fatty acids ratio, and fish consumption have a protective effect against cancer. On the contrary, a low intake of fruit and vegetables, high intake of red and processed meat, high intake of sodium, alcohol consumption, a diet rich in refined carbohydrates, and a high intake of total fat may increase risk of cancer. Furthermore, calorie restriction and having a body/mass index on the lower end of the normal range can significantly decrease or delay the onset of cancers. Most studies were performed on adults and thus the role of diet in childhood cancer is less well-understood. In the past, diet was not considered to play any role in its etiology in children. However, nowadays there is a growing body of evidence that prolonged and frequent breastfeeding, the maternal diet during pregnancy and vitamin intake during pregnancy, may impart benefit for reduced cancer risk in children. Usually, decades of healthy dietary habits are needed to see significant difference in cancer risk. Therefore, diet choices and diet preparation starting early in life deserve more attention. Here we review data focusing on which dietary factors, including food-borne carcinogens, affect the onset of cancers in adults and stress out the potential role of diet in childhood cancer prevention.

Synthesis, absolute configuration, and bacterial mutagenicity of the 8 stereoisomeric vicinal diol epoxides at the terminal benzo ring of carcinogenic dibenz[a,h]anthracene.

The synthesis of the 8 possible stereoisomeric diol epoxides (DEs) at the terminal benzo ring of carcinogenic dibenz[a,h]anthracene (DBA) is reported. trans-3,4-Dihydroxy-3,4-dihydro-DBA (1) afforded the 4 bay region DEs: the enantiomeric pairs of the anti diastereomers (+)-3/(-)-3 and of the syn diastereomers (-)-4/(+)-4, respectively. trans-1,2-Dihydroxy-1,2-dihydro-DBA (2) served as precursor of the 4 reverse DEs: the enantiomeric pairs of the anti diastereomers (+)-5/(-)-5 and of the syn diastereomers (-)-6/(+)-6, respectively. The transformation of the olefinic double bond in the enantiomeric trans-dihydrodiols to epoxides was achieved by either (i) oxidation with m-chloroperoxybenzoic acid or (ii) formation of a bromohydrin with N-bromoacetamide/H(2)O followed by dehydrobromination with an anion exchange resin. Because of the pseudodiequatorial conformation of the hydroxyl groups in 1, both reactions proceeded highly stereoselectively, while the stereoselectivity was impaired by the pseudodiaxial conformation of the hydroxyl groups in 2. Diastereomers and racemic compounds were efficiently separated without derivatization by HPLC on achiral or chiral stationary phases, respectively. The absolute configurations of the DEs were deduced from the absolute configuration of 1 and 2 considering the regio- and stereoselectivity of the subsequent reactions and resulted in (+)-(1R,2S,3S,4R)-3/(-)-(1S,2R,3R,4S)-3, (-)-(1S,2R,3S,4R)-4/(+)-(1R,2S,3R,4S)-4, (+)-(1R,2S,3S,4R)-5/(-)-(1S,2R,3R,4S)-5, and (-)-(1R,2S,3R,4S)-6/(+)-(1S,2R,3S,4R)-6. The bacterial mutagenicity of the 8 stereoisomeric DEs was determined in histidine-dependent strains TA98 and TA100 of Salmonella typhimurium in the absence of a metabolizing system. In general, the bay region DEs of DBA were stronger mutagens than the reverse DEs. In strain TA98, the syn diastereomers of bay region DEs were stronger mutagens than their anti isomers, while in the case of reverse DEs the anti diastereomers were more potent than their syn isomers. In strain TA100, all syn diastereomers surpassed the bacterial mutagenicity of their anti isomers. Concerning the bay region DEs of DBA, this corresponds to the situation described for benzo[a]pyrene: of the 4 enantiomeric bay region DEs of DBA and benzo[a]pyrene, the syn diastereomer with [(R,S)-diol (R,S)-epoxide] absolute configuration is the most potent mutagen in both bacterial strains, while the anti isomer with [(S,R)-diol (R,S)-epoxide] configuration is the weakest mutagen.

Influence of aryl hydrocarbon- (Ah) receptor and genotoxins on DNA repair gene expression and cell survival of mouse hepatoma cells.

The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called "bulky" DNA adducts including those generated by enzymatically activated B[a]P. Therefore, the hypothesis that AhR may enhance NER gene expression to trigger DNA repair in the presence of genotoxic AhR ligands was tested. Furthermore, we investigated a potential cytoprotective effect of AhR activation by the non-genotoxic ligand TCDD against cell death induced by various genotoxins. Finally, the actions of genotoxins themselves on NER gene expression were studied. As a cell culture model we used mouse hepatoma cells (Hepa-c7) proficient for AhR and its partner protein ARNT as well as subclones deficient in AhR (Hepa-c12) or ARNT (Hepa-c4) to study involvement of AhR and ARNT in response to B[a]P and TCDD. Indeed, the mRNA levels of the two NER genes XP-C and DNA polymerase kappa were increased by B[a]P and TCDD, however, this was not accompanied by an increase in the amount of the respective proteins. Pretreatment of cells with TCDD did not reduce cytotoxicity induced by various genotoxins. Thus, in Hepa-c7 cells AhR has no major effects on the expression of these crucial NER proteins and does not prevent genotoxin-provoked cell death. As expected, the genotoxins B[a]P and cis-platin led to p53 accumulation and induction of its target p21. Interestingly, however, NER gene expression was not enhanced but rather decreased. As two NER genes, XP-C and DNA damage binding protein ddb2, are up-regulated by p53 and ultraviolet radiation in human cells these findings suggest cell type, species or lesion specific actions of p53 on DNA repair gene expression. Importantly, in cells with damaged DNA up-regulation of p53 may not suffice to enhance DNA repair gene expression.

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region.

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.

Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions.

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[a]pyrene (BaP), benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11H-benzo[b]fluorene, dibenz[a,h]anthracene, pyrene, benzo[ghi]perylene and benzo[e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer. The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.

Microsomal biotransformation of benzo[ghi]perylene, a mutagenic polycyclic aromatic hydrocarbon without a "classic" bay region.

Carcinogenic polycyclic aromatic hydrocarbons (PAH), e.g., benzo[a]pyrene (BaP), possess a bay region comprising an ortho-fused benzene ring. Benzo[ghi]perylene (BghiP) represents the group of PAHs lacking such a "classic" bay region and hence cannot be metabolically converted like BaP to bay region dihydrodiol epoxides considered as ultimate mutagenic and carcinogenic metabolites of PAH. BghiP exhibits bacterial mutagenicity in strains TA98 (1.3 his(+)-revertant colonies/nmol) and TA100 (4.3 his(+)-revertant colonies/nmol) of Salmonella typhimurium after metabolic activation by the postmitochondrial hepatic fraction of CD rats treated with 3-methylcholanthrene. Inhibition of microsomal epoxide hydrolase (mEH) with 1,1,1-trichloro-2-propene oxide raised the bacterial mutagenicity of BghiP in TA98 almost 4-fold indicating arene oxides as ultimate mutagens. To confirm this assumption, the biotransformation of BghiP was elucidated. Incubation of BghiP with liver microsomes of CD rats treated with Aroclor 1254 yielded 17 ethyl acetate extractable metabolic products. Twelve metabolites were identified by a combination of chromatographic, spectroscopic, and biochemical methods. The microsomal biotransformation of BghiP proceeds by two pathways: Pathway I starts with the monooxygenase attack at the 7-position leading to the 7-phenol, which is transformed to the 7,8- and 7,10-diphenols followed by oxidation to the 7,8- and 7,10-quinones. On pathway II, the K regions of BghiP are successively converted to arene oxides yielding the indirectly identified 3,4-oxide and the 3,4,11,12-bisoxides. Enzymatic hydrolysis of the 3,4-oxide leads to the trans-3,4-dihydrodiol, which is oxidized to the 3,4-quinone. Similarly, the trans-3,4-trans-11,12-bisdihydrodiols and the trans-3,4-dihydrodiol 11,12-quinone are generated from the 3,4,11,12-bisoxides. The trans-3,4-dihydrodiol and the trans-3,4-trans-11,12-bisdihydrodiols are preferentially formed as R,R and R,R,R,R enantiomers, respectively. The intrinsic bacterial mutagenicity of the 3,4,11,12-bisoxides is rather low and hardly explains the strong increase in bacterial mutagenicity of BghiP after inhibition of mEH. Thus, we believe that the 3,4-oxide plays a more important role as the ultimate mutagenic metabolite of BghiP.

Tumor formation in the neonatal mouse bioassay indicates that the potent carcinogen dibenzo[def,p]chrysene (dibenzo[a,l]pyrene) is activated in vivo via its trans-11,12-dihydrodiol.

The hexacyclic aromatic hydrocarbon dibenzo[def,p]chrysene, better known as dibenzo[a,l]pyrene (DBP) in the field of chemical carcinogenesis, is present in the environment as a combustion product of organic matter. This compound is probably the strongest chemical carcinogen ever tested. As ultimate genotoxic metabolites of DBP two electrophilically reactive species are discussed: (i) radical cations generated by one-electron oxidation, and (ii) fjord region dihydrodiol epoxides formed via the trans-11,12-dihydroxy 11,12-dihydro derivative of DBP (11,12-dihydrodiol). In order to delineate the metabolic pathway(s) involved in tumor formation by DBP, newborn Crl:CD-1(ICR)BR mice were intraperitoneally treated with the parent compound, its 11,12-dihydrodiol, and the two diastereomeric fjord region dihydrodiol epoxides. Due to severe acute and chronic toxicity, the total dose of DBP and of the 11,12-dihydrodiol was limited to 40 nmol. For the same reason the dihydrodiol epoxides could only be applied in doses up to 0.4 nmol. The tumor incidence was determined 55 +/- 1 weeks after treatment. Under these conditions, DBP and its 11,12-dihydrodiol induced lung tumors (incidence: 86.5% versus 92.0%; yield: 2.88 versus 7.44 tumors per mouse), liver (incidence: 57.7% versus 60.0%; yield: 3.63 versus 5.28 tumors per mouse) and other organs (incidence: 36.5% versus 32.0%; yield: 0.56 versus 0.52 tumors per mouse). By contrast, only lung tumors at low incidence were detected in mice treated with solvent only (incidence: 28.8%; yield: 0.58 tumors per mouse). As with the parent hydrocarbon, mice treated with low doses of diastereomeric syn- and anti-dihydrodiol epoxides of DBP showed increased tumor incidences in liver (incidence: 19.0 and 46.7%; yield: 0.36 and 1.47 tumors per mouse, respectively), and in various other organs (incidence: 7.1 and 20.0%; yield: 0.07 and 0.20 tumors per mouse, respectively). In consideration of the 100-fold differences in the doses of compounds applied in this study, the tumor-inducing potency increases in the order DBP < 11,12-dihydrodiol < anti-dihydrodiol epoxide. This result provides strong evidence that the potent carcinogen DBP is activated in vivo in the mouse via its 11,12-dihydrodiol and not preferentially through alternative pathways.

Development and application of test methods for the detection of dietary constituents which protect against heterocyclic aromatic amines.

This article describes the development and use of assay models in vitro (genotoxicity assay with genetically engineered cells and human hepatoma (HepG2) cells) and in vivo (genotoxicity and short-term carcinogenicity assays with rodents) for the identification of dietary constituents which protect against the genotoxic and carcinogenic effects of heterocyclic aromatic amines (HAs). The use of genetically engineered cells expressing enzymes responsible for the bioactivation of HAs enables the detection of dietary factors that inhibit the metabolic activation of HAs. Human derived hepatoma (HepG2) cells are sensitive towards HAs and express several enzymes [glutathione S-transferase (GST), N-acetyltransferase (NAT), sulfotransferase (SULT), UDP-glucuronosyltransferase (UDPGT), and cytochrome P450 isozymes] involved in the biotransformation of HAs. Hence these cells may reflect protective effects, which are due to inhibition of activating enzymes and/or induction of detoxifying enzymes. The SCGE assay with rodent cells has the advantage that HA-induced DNA damage can be monitored in a variety of organs which are targets for tumor induction by HAs. ACF and GST-P(+) foci constitute preneoplastic lesions that may develop into tumors. Therefore, agents that prevent the formation of these lesions may be anticarcinogens. The foci yield and the sensitivity of the system could be substantially increased by using a modified diet. The predictive value of the different in vitro and in vivo assays described here for the identification of HA-protective dietary substances relevant for humans is probably better than that of conventional in vitro test methods with enzyme homogenates. Nevertheless, the new test methods are not without shortcomings and these issues are critically discussed in the present article.

Microsomal activation of dibenzo[def,mno]chrysene (anthanthrene), a hexacyclic aromatic hydrocarbon without a bay-region, to mutagenic metabolites.

Metabolically formed dihydrodiol epoxides in the bay-region of polycyclic aromatic hydrocarbons are thought to be responsible for the genotoxic properties of these environmental pollutants. The hexacyclic aromatic hydrocarbon dibenzo[def,mno]chrysene (anthanthrene), although lacking this structural feature, was found to exhibit considerable bacterial mutagenicity in histidine-dependent strains TA97, TA98, TA100, and TA104 of S. typhimurium in the range of 18-40 his(+)-revertant colonies/nmol after metabolic activation with the hepatic postmitochondrial fraction of Sprague-Dawley rats treated with Aroclor 1254. This mutagenic effect amounted to 44-84% of the values determined with benzo[a]pyrene under the same conditions. The specific mutagenicity of anthanthrene in strain TA100 obtained with the cell fraction of untreated animals was 6 his(+)-revertant colonies/nmol and increased 2.7-fold after treatment with phenobarbital and 4.5-fold after treatment with 3-methylcholanthrene. To elucidate the metabolic pathways leading to genotoxic metabolites, the microsomal biotransformation of anthanthrene was investigated. A combination of chromatographic, spectroscopic, and biochemical methods allowed the identification of the trans-4,5-dihydrodiol, 4,5-oxide, 4,5-, 1,6-, 3,6-, and 6,12-quinones, and 1- and 3-phenols. Furthermore, two diphenols derived from the 3-phenol, possibly the 3,6 and 3,9 positional isomers, as well as two phenol dihydrodiols were isolated. Three pathways of microsomal biotransformation of anthanthrene could be distinguished: The K-region metabolites are formed via pathway I dominated by monooxygenases of the P450 1B subfamily. On pathway II the polynuclear quinones of anthanthrene are formed. Pathway III is preferentially catalyzed by monooxygenases of the P450 1A subfamily and leads to the mono- and diphenols of anthanthrene. The K-region oxide and the 3-phenol are the only metabolites of anthanthrene with strong intrinsic mutagenicity, qualifying them as ultimate mutagens or their precursors. From the intrinsic mutagenicity of these two metabolites and their metabolic formation, the maximal mutagenic effect was calculated. This demonstrates the dominating role of pathway III in the mutagenicity of anthanthrene under conditions where it exhibits the strongest bacterial mutagenicity.