3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.     

Biotransformation of xenobiotics in the human colon and rectum and its association with colorectal cancer

In humans, the liver is generally considered to be the major organ contributing to drug metabolism, but studies during the last years have suggested an important role of the extra-hepatic drug metabolism. The gastrointestinal tract (GI-tract) is the major path of entry for a wide variety of compounds including food, and orally administered drugs, but also compounds – with neither nutrient nor other functional value – such as carcinogens. These compounds are metabolized by a large number of enzymes, including the cytochrome P450 (CYP), the glutathione S-transferase (GST) family, the uridine 5′-diphospho- glucuronosyltransferase (UDP-glucuronosyltransferase – UGT) superfamily, alcohol-metabolizing enzymes, sulfotransferases, etc. These enzymes can either inactivate carcinogens or, in some cases, generate reactive species with higher reactivity compared to the original compound. Most data in this field of research originate from animal or in vitro studies, wherein human studies are limited. Here, we review the human studies, in particular the studies on the phenotypic expression of these enzymes in the colon and rectum to get an impression of the actual enzyme levels in this primary organ of exposure. The aim of this review is to give a summary of currently available data on the relation between the CYP, the GST and the UGT biotransformation system and colorectal cancer obtained from clinical and epidemiological studies in humans.     

Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling analysis using different chromatographic procedures

We report the analysis of DNA adducts in the target organ (forestomach)of male Sprague–Dawley rats treated orally with two doses(10 mg/kg body wt) per week for 2 weeks of either aristolochicacid I (AAI), aristolochic acid II (AAII) or the plant extractaristolochic acid (AA). DNA adducts were detected and quantitatedusing the nuclease P1-enhanced version of the ³²P-postlabellingassay. For identification of adducts, reference compounds wereprepared by reaction of enzymatically activated AAI and AAIIwith 3'-purine phosphonucleosides and analysed by the ⁿ-butanolenrichment procedure. These reference compounds were assignedto the previously characterized DNA adducts of AAI [7-(deoxy-guanosin-N²-yl)-aristolactamI = dG-AAI, 7-(deoxyadenosin-N⁶-yl) I = dA-AAI] and AAII [7-(deoxyadenosin-N⁶-yl)-aristolactamII = dA-AAII]. Cross referencing of the carcinogen-modifiednucleoside bisphosphates obtained from forestomach DNA withthe synthetic standard compounds by ion-exchange chromatographyand reversed-phase HPLC demonstrated that the major DNA adductsformed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise,forestomach DNA isolated from AAII-treated rats showed two purinederived adduct spots, the major one being dA-AAII, the minorone being tentatively identified as 7-(deoxyguanosin-N²-yl)-aristolactamII. A minor adduct detected in forestomach DNA of rats treatedwith AAI was found to be chromatographically indistinguishablefrom the adduct identified as dA-AAII, indicating a possibledemethoxylation reaction of AAI. Quantitation of DNA adductsrevealed that in in vitro reactions with 3'-phosphonucleosidesthe adduct levels were approximately one order higher for bothAAI and AAII-derived adducts than in forestomach DNA modifiedwith AAI or AAII in vivo. In vitro as well as in vivo adductionby AAI was more efficient than adduction by AAII. The patternof adduct spots obtained from forestomach DNA of rats treatedwith the plant extract AA reflected the composition of the extractdetermined by HPLC analysis. Irrespective of the aristolochicacid used to induce DNA adducts, deoxyadenosine is the majortarget of modification, pointing to the general importance ofdeoxyadenosine adducts for chemical carcinogenesis of thesenaturally occurring products. This study shows that the combinationof two independent chromatographic systems considerably enhancesthe fidelity of identification of DNA adducts with the ³²P-posthbellingassay.     

Environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosols

3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the (32)P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,O-acetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10- to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs.     

Aristolochic acid as a probable human cancer hazard in herbal remedies: a review

The old herbal drug aristolochic acid (AA), derived from Aristolochia spp., has been associated with the development of a novel nephropathy, designated aristolochic acid nephropathy (AAN), and urothelial cancer in AAN patients. There is clear evidence that the major components of the plant extract AA, aristolochic acid I (AAI) and aristolochic acid II (AAII), both nitrophenanthrene carboxylic acids, are genotoxic mutagens forming DNA adducts after metabolic activation through simple reduction of the nitro group. Several mammalian enzymes have been shown to be capable of activating both AAI and AAII in vitro and in cells. The activating metabolism has been elucidated and is consistent with the formation of a cyclic nitrenium ion with delocalized charge leading to the preferential formation of purine adducts bound to the exocyclic amino groups of deoxyadenosine and deoxyguanosine. The predominant DNA adduct in vivo, 7-(deoxyadenosin-N(6)-yl)aristolactam I (dA-AAI), which is the most persistent of the adducts in target tissue, is a mutagenic lesion leading to AT-->TA transversions in vitro. This transversion mutation is found at high frequency in codon 61 of the H-ras oncogene in tumours of rodents induced by AAI, suggesting that dA-AAI might be the critical lesion in the carcinogenic process in rodents. DNA-binding studies confirmed that both AAs bind to the adenines of codon 61 in the H-ras mouse gene and preferentially to purines in the human p53 gene. In contrast, the molecular mechanism of renal interstitial fibrosis in humans after chronic administration of AA remains to be explored. However, preliminary findings suggest that DNA damage by AA is not only responsible for the tumour development but also for the destructive fibrotic process in the kidney. It is concluded that there is significant evidence that AA is a powerful nephrotoxic and carcinogenic substance with an extremely short latency period, not only in animals but also in humans. In particular, the highly similar metabolic pathway of activation and resultant DNA adducts of AA allows the extrapolation of carcinogenesis data from laboratory animals to the human situation. Therefore, all products containing botanicals known to or suspected of containing AA should be banned from the market world wide.     

Neuroblastoma stem cells - mechanisms of chemoresistance and histonedeacetylase inhibitors

Cancer stem cells (CSCs) form a?small proportion of tumor cells that have stem cell properties: self-renewal capacity, the ability to develop into different lineages and proliferative potential. The interest in CSCs emerged from their expected role in initiation, progression and recurrence of many tumors. They are generally resistant to conventional chemotherapy and radiotherapy. There are two hypotheses about their origin: The first assumes that CSCs may arise from normal stem cells, and the second supposes that differentiated cells acquire the properties of CSCs. Both hypotheses are not mutually exclusive, as it is possible that CSCs have a?diverse origin in different tumors. CD133+ cells (CD133 is marker of CSC in some tumors) isolated from NBL, osteosarcoma and Ewing sarcoma cell lines are resistant to cisplatin, carboplatin, etoposide and doxorubicin than the CD133- ones. Being resistant to chemotherapy, there were many attempts to target CSCs epigenetically including the use of histone deacetylase inhibitors. The diverse influence of valproic acid (histone deacetylase inhibitor) on normal and cancer stem cells was proved in different experiments. We have found an increase percentage of CD133+ NBL cells after their incubation with VPA in a?dose that does not induce apoptosis. Further researches on CSCs and clinical application for their detection are necessary: (i) to define the CSC function in carcinogenesis, cancer development and their role in metastasis; (ii) to find a?specific marker for CSCs in different tumors; (iii) to explain the role of different pathways that determine their behavior and (iv) to explain mechanisms of chemoresistance of CSCs. Keywords: Cancer stem cells; CD133; Neuroblastoma; Histone deacetylase inhibitors.     

Exceptionally long‐term persistence of DNA adducts formed by carcinogenic aristolochic acid I in renal tissue from patients with aristolochic acid nephropathy

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7‐(deoxyadenosin‐N⁶‐yl)‐aristolactam I (dA‐AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA‐AAI adduct induces AT to TA transversions in the tumor‐suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin‐layer chromatography ³²P‐postlabeling and mass spectrometric analysis we report the detection of dA‐AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA‐AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA‐AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA‐associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long‐term persistence of dA‐AAI provides a useful long‐term biomarker of exposure and attests to the role of AA in human urothelial malignancy.     

Anthracyclines and ellipticines as DNA-damaging anticancer drugs: recent advances

Over the past forty years, anthracyclines and ellipticines have attracted attention as promising cytostatics. In this review, we focus on their mechanisms of cytoxicity, DNA-damaging effects and adverse side-effects. We also summarize ways to enhance the therapeutic effects of these drugs together with a decrease in their adverse effects. Current drug design strategies are focused on drug bioavailability and their tissue targeting, whereas drug delivery to specific intracellular compartments is rarely addressed. Therefore, therapies utilizing the antineoplastic activities of anthracyclines and ellipticines combined with novel strategies such as nanotechnologies for safer drug delivery, as well as strategies based on gene therapy, could significantly contribute to medical practice.