Genotoxicity studies with mineral oils; effects of oils on the microbial mutagenicity of precursor mutagens and genotoxic metabolites

In vitro genotoxicity assays are extensively used to predict carcinogenic activity in vivo. The standard microbial mutagenicity assays however often fail to yield positive results with mineral oils which are carcinogenic to mice in long-term skin-cancer studies. A comprehensive programme of studies has therefore investigated the basis of this apparently anomalous behaviour. This investigation has addressed the possible effects of oils on the bioactivation of precursor mutagens and the disposition of mutagenic metabolites by studying the microbial mutagenicity of selected precursor mutagens (benzo[a]pyrene, benzo[a]anthracene, 2-aminoanthracene and 2-naphthylamine) and intrinsically reactive mutagens [+/- )-benzo[a]pyrene-4,5-oxide and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) in the presence and absence of mineral oils. Notably the mutagenicity associated with the deliberate additions of these mutagens or precursor mutagens to oils was readily detected by the microbial assays. The mutagenicity of only one of the precursor mutagens, benzo[a]pyrene, was significantly reduced by the oils, and then only in the standard plate-incorporation assay. Interestingly the degree of suppression appeared to be related to the polycyclic aromatic hydrocarbon content of the oils. In the case of 2-aminoanthracene large enhancements in its mutagenicity were observed in the presence of oils. These latter findings appear to be due to effects of oils on the bioactivation of precursor mutagens rather than on the disposition of their bioactivation products. The mutagenicity of intrinsically reactive mutagens, of a type generated by bioactivation of polycyclic aromatic hydrocarbons, was not significantly reduced in the presence of mineral oils. This indicates that it is unlikely that components in oils trap or facilitate the deactivation of ultimate mutagens whether these pre-exist in the oil or are formed from precursors by bioactivation in the in vitro test system. Viewed overall these results suggest that mineral oils judged to be carcinogenic on the basis of in vivo studies in mouse skin may possess only very weak genotoxic potential. While this potential is likely to be a prerequisite for carcinogenic action, the current results cause attention to be focussed on other factors, e.g. promotion, as potentially important determinants of the carcinogenic potencies of mineral oils in mouse skin.     

Genotoxicity and mutagenicity of chromium(VI)/ascorbate-generated DNA adducts in human and bacterial cells

Reduction of carcinogenic Cr(VI) by vitamin C generates ascorbate-Cr(III)-DNA cross-links, binary Cr(III)-DNA adducts, and can potentially cause oxidative DNA damage by intermediate reaction products. Here, we examined the mutational spectrum and the importance of different forms of DNA damage in genotoxicity and mutagenicity of Cr(VI) activated by physiological concentrations of ascorbate. Reduction of Cr(VI) led to a dose-dependent formation of both mutagenic and replication-blocking DNA lesions as detected by propagation of the pSP189 plasmids in human fibroblasts. Disruption of Cr-DNA binding abolished mutagenic responses and normalized the yield of replicated plasmids, indicating that Cr-DNA adducts were responsible for both mutagenicity and genotoxicity of Cr(VI). The absence of DNA breaks and abasic sites confirmed the lack of a significant production of hydroxyl radicals and Cr(V)-peroxo complexes in Cr(VI)-ascorbate reactions. Ascorbate-Cr(III)-DNA cross-links were much more mutagenic than smaller Cr(III)-DNA adducts and accounted for more than 90% of Cr(VI) mutagenicity. Ternary adducts were also several times more potent in the inhibition of replication than binary complexes. The Cr(VI)-induced mutational spectrum consisted of an approximately equal number of deletions and G/C-targeted point mutations (51% G/C --> T/A and 30% G/C --> A/T). In Escherichia coli cells, Cr(VI)-induced DNA adducts were only highly genotoxic but not mutagenic under either normal or SOS-induced conditions. Lower toxicity and high mutagenicity of ascorbate-Cr(III)-DNA adducts in human cells may result from the recruitment of an error-prone bypass DNA polymerase(s) to the stalled replication forks. Our results suggest that phosphotriester-type DNA adducts could play a more important role in human than bacterial mutagenesis.     

Carcinogenic chromium(VI) induces cross-linking of vitamin C to DNA in vitro and in human lung A549 cells

Reductive activation of carcinogenic Cr(VI) is required for the induction of DNA damage and mutations. Here, we examined the formation of Cr-DNA adducts in the reactions of Cr(VI) with its dominant biological reducer, vitamin C (ascorbate). Reductive conversion of Cr(VI) to Cr(III) by ascorbate produced stable Cr-DNA adducts, of which approximately 25% constituted ascorbate-Cr(III)-DNA cross-links. No evidence was found for the involvement of Cr(V) or Cr(IV) intermediates in the formation of either binary or ternary adducts. The cross-linking reaction was consistent with the attack of DNA by transient Cr(III)-ascorbate complexes. The yield of Cr(III)-DNA adducts was similar on dsDNA and AGT, ACT, or CT oligonucleotides and was strongly inhibited by Mg(2+), suggesting predominant coordination of Cr(III) to DNA phosphate oxygens. We also detected cross-linking of ascorbate to DNA in Cr(VI)-exposed human lung A549 cells that were preincubated with dehydroascorbic acid to create normal levels of intracellular ascorbate. Ascorbate-Cr-DNA cross-links accounted for approximately 6% of the total Cr-DNA adducts in A549 cells. Shuttle-vector experiments showed that ascorbate-Cr-DNA cross-links were mutagenic in human cells. Our results demonstrate that in addition to reduction of Cr(VI) to DNA-reactive Cr(III), vitamin C contributes to the genotoxicity of Cr(VI) via a direct chemical modification of DNA. The absence of Asc in A549 and other human cultured cells indicates that cells maintained under the usual in vitro conditions lack the most important reducing agent for Cr(VI) and would primarily display slow thiol-dependent activation of Cr(VI).     

Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents

Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.     

Genotoxicity of quinone pigments from pathogenic fungi

The genotoxicity and mutagenicity of several kinds of quinone pigments from pathogenic fungi were examined by means of the hepatocyte primary culture (HPC)/DNA repair test and of Ames test with TA98 and TA100. Clear genotoxicity of the two quinone chemicals, xanthomegnin and luteosporin were observed in the HPC/DNA repair test, though definite mutagenicity was not detected in the Salmonella microsome test. These two pigments are thus suspected to be genotoxic carcinogens.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.     

Identification of o-quinone/quinone methide metabolites of quercetin in a cellular in vitro system

Formation of quercetin quinone/quinone methide metabolites, reflected by formation of the glutathionyl quercetin adducts as authentic metabolites, was investigated in an in vitro cell model (B16F-10 melanoma cells). Results of the present study clearly indicate the formation of glutathionyl quercetin adducts in a tyrosinase-containing melanoma cell line, expected to be representative also for peroxidase-containing mammalian cells and tissues. The data obtained also support that the adducts are formed intracellular and subsequently excreted into the incubation medium and reveal for the first time evidence for the pro-oxidative metabolism of quercetin in a cellular in vitro model.     

Structure—activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK_a′ = 4.2–4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4′-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4–8 times the background were observed at the higher doses (1000 μg/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.     

Genotoxicity of environmental tobacco smoke: a review

Environmental tobacco smoke (ETS), or second-hand smoke, is a widespread contaminant of indoor air in environments where smoking is not prohibited. It is a significant source of exposure to a large number of substances known to be hazardous to human health. Numerous expert panels have concluded that there is sufficient evidence to classify involuntary smoking (or passive smoking) as carcinogenic to humans. According to the recent evaluation by the International Agency for Research on Cancer, involuntary smoking causes lung cancer in never-smokers with an excess risk in the order of 20% for women and 30% for men. The present paper reviews studies on genotoxicity and related endpoints carried out on ETS since the mid-1980s. The evidence from in vitro studies demonstrates induction of DNA strand breaks, formation of DNA adducts, mutagenicity in bacterial assays and cytogenetic effects. In vivo experiments in rodents have shown that exposure to tobacco smoke, whole-body exposure to mainstream smoke (MS), sidestream smoke (SS), or their mixture, causes DNA single strand breaks, aromatic adducts and oxidative damage to DNA, chromosome aberrations and micronuclei. Genotoxicity of transplacental exposure to ETS has also been reported. Review of human biomarker studies conducted among non-smokers with involuntary exposure to tobacco smoke indicates presence of DNA adducts, urinary metabolites of carcinogens, urinary mutagenicity, SCEs and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutations (in newborns exposed through involuntary smoking of the mother). Studies on human lung cancer from smokers and never-smokers involuntarily exposed to tobacco smoke suggest occurrence of similar kinds of genetic alterations in both groups. In conclusion, these overwhelming data are compatible with the current knowledge on the mechanisms of carcinogenesis of tobacco-related cancers, occurring not only in smokers but with a high biological plausibility also in involuntary smokers.     

Metabolomic Profiling Unravels DNA Adducts in Human Breast That Are Formed from Peroxidase Mediated Activation of Estrogens to Quinone Methides

Currently there are three major hypotheses that have been proposed for estrogen induced carcinogenicity, however exact etiology remains unknown. Based on the chemical logic, studies were undertaken to investigate if estrogens could generate quinone methides in an oxidative environment which then could cause DNA damage in humans. In presence of MnO₂ estrogens were oxidized to quinone methides. Surprisingly quinone methides were found to be stable with t_1/2 of 20.8 and 4.5 min respectively. Incubation of estrogens with lactoperoxidase (LPO) and H₂O₂ resulted in formation of respective quinone methides (E₁(E₂)-QM). Subsequent addition of adenine to the assay mixture lead to trapping of E₁(E₂)-QM, resulting in formation of adenine adducts of estrogens, E₁(E₂)-9-N-Ade. Targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based metabolomic analysis of the breast tissue extracts showed the presence of adenine adducts of estrogens, E₁(E₂)-9-N-Ade, along with other estrogen related metabolites. Identity of E₁(E₂)-N-Ade in LPO assay extracts and breast tissue extracts were confirmed by comparing them to pure synthesized E₁(E₂)-9-N-Ade standards. From these results, it is evident that peroxidase enzymes or peroxidase-like activity in human breast tissue could oxidize estrogens to electrophilic and stable quinone methides in a single step that covalently bind to DNA to form adducts. The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer. This article reports evidence for hitherto unknown estrogen metabolic pathway in human breast, catalyzed by peroxidase, which could initiate cancer.     

Herbal bioactivation, molecular targets and the toxicity relevance

There have been increasing reports on the adverse reactions associated with herbal consumption. For many of these adverse reactions, the underlying biochemical mechanisms are unknown, but bioactivation of herbal compounds to generate reactive intermediates have been implicated. This minireview updates our knowledge on metabolic activation of herbal compounds, molecular targets and the toxicity relevance. A number of studies have documented that some herbal compounds can be converted to toxic or even carcinogenic metabolites by Phase I [e.g. cytochrome P450s (CYPs)] and less frequently by Phase II enzymes. For example, aristolochic acids (AAs) in Aristolochia spp, which undergo reduction of the nitro group by hepatic CYP1A1/2 or peroxidases in extrahepatic tissues to generate highly reactive cyclic nitrenium ions. The latter can react with macromolecules (DNA and protein), resulting in activation of H-ras and myc oncogenes and gene mutation in renal cells and finally carcinogenesis of the kidneys. Teucrin A and teuchamaedryn A, two diterpenoids found in germander (Teuchrium chamaedrys) used as an adjuvant to slimming herbal supplements that caused severe hepatotoxicity, are converted by CYP3A4 to reactive epoxide which reacts with proteins such as CYP3A and epoxide hydrolase and inactivate them. Some naturally occurring alkenylbenzenes (e.g. safrole, methyleugenol and estragole) and flavonoids (e.g. quercetin) can undergo bioactivation by sequential 1-hydroxylation and sulfation, resulting in reactive intermediates capable of forming DNA adducts. Extensive pulegone metabolism generated p-cresol that is a glutathione depletory. The hepatotoxicity of kava is possibly due to intracellular glutathione depletion and/or quinone formation. Moreover, several herbal compounds including capsaicin from chili peppers, dially sulfone in garlic, methysticin and dihydromethysticin in kava, oleuropein in olive oil, and resveratrol found in grape seeds are mechanism-based (suicide) inhibitors of various CYPs. Together with advances of proteomics, metabolomics and toxicogenomics, an integrated systems toxicological approach may provide deep insights into mechanistic aspects of herb-induced toxicities, and contribute to bridging the relationships between herbal bioactivation, protein/DNA adduct formation and the toxicological consequences.     

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.     

Studies on biomarkers in cancer etiology and prevention: a summary and challenge of 20 years of interdisciplinary research

Sensitive, specific methods have been developed that allow quantitative measurements of the metabolites of carcinogen metabolites and of DNA and protein adducts in humans exposed occupationally, environmentally and endogenously to genotoxic agents. The interrelationship between exposure to carcinogens, host risk factors and the responses of biomarkers has been examined in cross-sectional, ecological and case-control studies which provided new insights into the causes of cancer and the mechanisms of carcinogenesis. The identification of hitherto unknown DNA-reactive chemicals formed in the human body from dietary precursors and of carcinogenic components of complex mixtures has increased the possibility of establishing causal relationships in etiology. The identification of individuals and subgroups heavily exposed to carcinogens has led to the development of measures for avoiding or decreasing exposure to carcinogenic risk factors. New, ultrasensitive methods for measuring DNA adducts allow the quantification and structural elucidation of specific DNA damage in humans arising from oxidative stress and lipid peroxidation (LPO), which have been found to be the driving forces in several human malignancies. Background DNA damage in "unexposed" individuals has been shown unequivocally to be due to LPO products, and a significant interindividual variation in adduct levels has been shown in individuals with comparable exposure to carcinogens. Thus, pharmacogenetic variants with higher susceptibility to carcinogenic insults, due to genetic polymorphism in xenobiotic-metabolizing enzymes, have been characterized by a combination of genotyping and measurements of macromolecular adducts. Dosimetry has been used in human studies to evaluate the efficacy of interventions with chemopreventive agents like ascorbic acid, dietary phenols and green tea. Advances in the application of selected biomarkers in human studies are reviewed and illustrated by examples from the author's research conducted during the past two decades.     

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.     

Inflammatory and genotoxic effects of diesel particles in vitro and in vivo

Recent studies have identified an indirect genotoxicity pathway involving inflammation as one of the mechanisms underlying the carcinogenic effects of air pollution/diesel exhaust particles (DEP). We investigated the short-term effects of DEP on markers of inflammation and genotoxicity in vitro and in vivo. DEP induced an increase in the mRNA level of pro-inflammatory cytokines and a higher level of DNA strand breaks in the human lung epithelial cell line A549 in vitro. For the in vivo study, mice were exposed by inhalation to 20 or 80 mg/m3 DEP either as a single 90-min exposure or as four repeated 90-min exposures (5 or 20 mg/m3) and the effects in broncho-alveolar lavage (BAL) cells and/or lung tissue were characterized. Inhalation of DEP induced a dose-dependent inflammatory response with infiltration of macrophages and neutrophils and elevated gene expression of IL-6 in the lungs of mice. The inflammatory response was accompanied by DNA strand breaks in BAL cells and oxidative DNA damage and increased levels of bulky DNA adducts in lung tissue, the latter indicative of direct genotoxicity. The effect of a large single dose of DEP was more pronounced and sustained on IL-6 expression and oxidative DNA damage in the lung tissue than the effect of the same dose administered over four days, whereas the reverse pattern was seen in BAL cells. Our results suggest that the effects of DEP depend on the rate of delivery of the particle dose. The mutation frequency (MF), after DEP exposure, was determined using the transgenic Muta Mouse and a similar exposure regimen. No increase was observed in MF in lung tissue 28-days after exposure. In conclusion, short-term exposure to DEP resulted in DNA strand breaks in BAL cells, oxidative DNA damage and DNA adducts in lungs; and suggested that DNA damage in part is a consequence of inflammatory processes. The response was not associated with increased MF, indicating that the host defence mechanisms were sufficient to counteract the adverse effects of inflammation. Thus, there may be thresholds for the inflammation-associated genotoxic effects of DEP inhalation.     

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA adducts derived from administration of acrylamide and glycidamide to mice and rats

Acrylamide (AA) is an important industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in chronic rodent bioassays. Recent findings of AA in many common starchy foods have sparked renewed interest in determining toxic mechanisms and in understanding the cancer, neurotoxicity, and reproductive risks from typical human exposures. Dosing mice and rats with AA (50 mg/kg) led to presence of glycidamide (GA) in serum and tissues. Furthermore, GA-derived DNA adducts of adenine and guanine were formed in all tissues examined, including both target tissues identified in rodent carcinogenicity bioassays and in non-target tissues. Dosing rats and mice with an equimolar amount of GA typically produced higher levels of DNA adducts than observed with AA. Kinetics of DNA adduct formation and accumulation were measured following oral administration of a single dose of AA (50 mg/kg) or from repeat dosing (1 mg/kg/day), respectively. The formation of these DNA adducts is consistent with previously reported mutagenicity of AA and GA in vitro, which involved reaction of GA with adenine and guanine bases. These results provide strong support for a genotoxic mechanism of AA carcinogenicity in rodents. The kinetic/biomarker approaches described here may represent a meaningful way to extrapolate cancer risks to actual human exposures from food, which are much lower.     

DNA adducts derived from administration of acrylamide and glycidamide to mice and rats

Acrylamide (AA) is an important industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in chronic rodent bioassays. Recent findings of AA in many common starchy foods have sparked renewed interest in determining toxic mechanisms and in understanding the cancer, neurotoxicity, and reproductive risks from typical human exposures. Dosing mice and rats with AA (50 mg/kg) led to presence of glycidamide (GA) in serum and tissues. Furthermore, GA-derived DNA adducts of adenine and guanine were formed in all tissues examined, including both target tissues identified in rodent carcinogenicity bioassays and in non-target tissues. Dosing rats and mice with an equimolar amount of GA typically produced higher levels of DNA adducts than observed with AA. Kinetics of DNA adduct formation and accumulation were measured following oral administration of a single dose of AA (50 mg/kg) or from repeat dosing (1 mg/kg/day), respectively. The formation of these DNA adducts is consistent with previously reported mutagenicity of AA and GA in vitro, which involved reaction of GA with adenine and guanine bases. These results provide strong support for a genotoxic mechanism of AA carcinogenicity in rodents. The kinetic/biomarker approaches described here may represent a meaningful way to extrapolate cancer risks to actual human exposures from food, which are much lower.     

Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Safrole–DNA adducts in tissues from esophageal cancer patients: clues to areca-related esophageal carcinogenesis

Epidemiological studies have demonstrated that areca quid chewing can be an independent risk factor for developing esophageal cancer. However, no studies are available to elucidate the mechanisms of how areca induces carcinogenesis in the esophagus. Since the areca nut in Taiwan contains a high concentration of safrole, a well-known carcinogenic agent, we analyzed safrole–DNA adducts by the ³²P-postlabelling method in tissue specimens from esophageal cancer patients. In total, we evaluated 47 patients with esophageal cancer (16 areca chewers and 31 non-chewers) who underwent esophagectomy at the National Taiwan University Hospital between 1996 and 2002. Of the individuals with a history of habitual areca chewing (14 cigarette smokers and two non-smokers), one of the tumor tissue samples and five of the normal esophageal mucosa samples were positive for safrole–DNA adducts. All patients positive for safrole–DNA adducts were also cigarette smokers. Such adducts could not be found in patients who did not chew areca, irrespective of their habits of alcohol consumption or cigarette smoking (p < 0.001, comparing the areca chewers with non-chewers). The genotoxicity of safrole was also tested in vitro in three esophageal cell lines and four cultures of primary esophageal keratinocytes. In two of the esophageal keratinocyte cultures, adduct formation was increased by treatment with safrole after induction of cytochrome P450 by 3-methyl-cholanthrene. This paper provides the first observation of how areca induces esophageal carcinogenesis, i.e., through the genotoxicity of safrole, a component of the areca juice.