DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay

The genotoxic activities of complex mixtures of organic extracts from the urban air particles collected in various localities of the Czech Republic, which differed in the extent and sources of air pollution, were compared. For this purpose, PM2.5 particles were collected by high volume samplers in the most polluted area of the Czech Republic - Ostrava region (localities Bartovice, Poruba and Karvina) and in the locality exhibiting a low level of air pollution - Trebon - a small town in the non-industrial region of Southern Bohemia. To prepare extractable organic matter (EOM), PM2.5 particles were extracted by dichloromethane and c-PAHs contents in the EOMs were determined. As markers of genotoxic potential, DNA adduct levels and oxidative DNA damage (8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxodG, levels) induced by EOMs in an acellular assay of calf thymus DNA coupled with ³²P-postlabeling (DNA adducts) and ELISA (8-oxodG) in the presence and absence of microsomal S9 fraction were employed. Twofold higher DNA adduct levels (17.20 adducts/10⁸ nucleotides/m³ vs. 8.49 adducts/10⁸ nucleotides/m³) were induced by EOM from Ostrava-Bartovice (immediate proximity of heavy industry) compared with that from Ostrava-Poruba (mostly traffic emissions). Oxidative DNA damage induced by EOM from Ostrava-Bartovice was more than fourfold higher than damage induced by EOM from Trebon (8-oxodG/10⁸ dG/m³: 0.131 vs. 0.030 for Ostrava-Bartovice vs. Trebon, respectively). Since PM2.5 particles collected in various localities differ with respect to their c-PAHs content, and c-PAHs significantly contribute to genotoxicity (DNA adduct levels), we suggest that monitoring of PM2.5 levels is not a sufficient basis to assess genotoxicity of respirable aerosols. It seems likely that the industrial emissions prevailing in Ostrava-Bartovice represent a substantially higher genotoxic risk than mostly traffic-related emissions in Ostrava-Poruba. B[a]P and c-PAH contents in EOMs are the most important factors relating to their genotoxic potential.     

An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part I: DNA adducts

An acellular assay consisting of calf thymus DNA with/without rat liver microsomal S9 fraction was used to study the genotoxicity of complex mixtures of organic air pollutants bound to size segregated aerosols by means of DNA adduct analysis. We compared the genotoxicity of the organic extracts (EOMs) from three size fractions of aerosol ranging from 0.17 μm to 10 μm that were collected by high volume cascade impactors in four localities of the Czech Republic differing in the extent of the environmental pollution: (1) small village in proximity of a strip mine, (2) highway, (3) city center of Prague and (4) background station. The total DNA adduct levels induced by 100 μg/ml of EOMs were analyzed by ³²P-postlabelling analysis with a nuclease P1 method for adduct enrichment. The main finding of the study was most of the observed genotoxicity was connected with a fine particulate matter fraction (<1 μm). The concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in EOMs indicate that fine fractions (0.5–1 μm) bound the highest amount of c-PAHs in all aerosol sampling sites, which might be related to the higher specific surface of this fraction as compared with a course fraction (1–10 μm) and higher mass as compared with a condensational fraction (0.17–0.5 μm). As for aerosol mass, both fine and condensational fractions are effective carriers of c-PAHs. Similarly, the DNA adduct levels per m³ of air were highest for the fine fraction, while the condensational fraction (strip mine site and city center) revealed the highest DNA adduct levels in cases where aerosol mass is taken into consideration. A strong correlation was found between the c-PAHs and DNA adduct levels induced by EOMs in all the localities and for various size fractions (R² = 0.98, p < 0.001). It may be concluded that the analysis of total DNA adducts induced in an acellular assay with/without metabolic activation represents a relatively simple method to assess the genotoxic potential of various complex mixtures.     

Influence of red blood cells,serum albumin,and serum lipoproteins on the clearance of benzo[a]pyrene by isolated livers of 3-methylcholanthrene-treated rats

Red blood cells, serum albumin, and serum lipoproteins transport benzo[a]pyrene and other xenobiotic compounds in the circulation. The distribution of benzo[a]pyrene and its metabolites among these blood components was examined, and the effect of their presence in the perfusion medium on the ability of isolated livers from 3-methylcholanthrene-pretreated rats to clear circulating benzo[a]pyrene was determined. A large fraction (45%) of the benzo[a]pyrene in rat blood was associated with the serum lipoproteins. However, only 8% of the benzo[a]pyrene metabolites was associated with this component. Forty to forty-five percent of each was associated with red blood cells. Benzo[a]pyrene clearance by isolated rat livers was 1.8 ± 0.2 ml/min when the medium contained only red blood cells and buffer. Addition of serum lipoproteins or serum albumin increased benzo[a]pyrene clearance to 5.1 ± 0.5 or 8.5 ± 0.9 ml/min respectively. Appearance of benzo[a]pyrene metabolites in perfusion medium and bile was similarly altered by the changes in medium composition. These results indicate that the clearance of benzo[a]pyrene by rat liver depends on the composition of the medium perfusing the organ and suggest that alterations in blood components in vivo may influence the metabolic disposition of this carcinogen.     

Effect of carcinogenic polycyclic aromatic hydrocarbons on mouse embryonic cells in culture: Induction of spindle-shaped cells

In cultured mouse embroyonic cells (MECs) treated with benzo[a]pyrene(B[a]P), there appeared unusual type of fibroblasts, spindle-shaped cells (SP cells), which were characterized by their narrow bipolar shape, long cellular processes and optically distinct cell borders. Appearance of SP cells was massive and irreversible. The amount of SP cells increased with increased with increasing concentrations of B[a]P, while early cytotoxicity did not. In various polycyclic aromatic hydrocarbons (PAHs) tested, only potent carcinogens {7,12-dimethylabenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), B[a]P and dibenz[a, e]pyrene (DB[a, e]P)} induced SP cells. Among them, PAH having higher Iball's index induced SP cells at lower concentration and at an earlier time. Weak or non-carcinogenic PAHs including 3-hydroxybenzo-[a]-pyrene(3-H-B[a]P) did not induce SP cells. α-Napthoflavon (αNF) suppressed the induction of SP cell by carcinogenic PAH. SP cells did not appear spontaneously under various abnormal culture conditions. These results indicate that carcinogenic PAHs induce the appearance of a specific type of fibroblast, SP cells in MEC cultures in accordance with their carcinogenicity.     

Transformation of BHK 21/CL 13 cells by various polycyclic aromatic hydrocarbons using the method of styles

Nine polycyclic aromatic hydrocarbons (PAHs) were investigated by the cell-transformation assay method of Styles. Benzo(a)pyrene [B(a)P], chrysene (CH), 3-methylcholanthrene (3-MC), benz(a)anthracene (BA), benzo(b)fluoranthene [B(b)F], and dibenz(a,h)anthracene (DBA) were tested, including liver homogenate, and showed dose-effect relationships and a more than 2-fold increase of transformation rates at LC₅₀. Due to variations of the test method our results differed quantitatively from the data published by Purchase and Styles. Discrimination between the known carcinogens listed above and the noncarcinogens, phenanthrene (PA) and anthracene (AC), lacking a dose-effect relationship was, however, possible. Benzo(e)pyrene [B(e)P] was regarded as positive although producing only a 2-fold increase in the number of transformed colonies.     

Effects of microsomal enzyme inducers in vivo and inhibitors in vitro on the covalent binding of benzo[a]pyrene metabolites to DNA catalyzed by liver microsomes from genetically responsive and nonresponsive mice.

The in vitro DNA binding of benzo[a]pyrene metabolites generated by mouse liver microsomes can be resolved into at least nine distinct peaks by elution of a Sephadex LH20 column with a water-methanol gradient. These peaks, representing metabolite-nucleoside complexes, are named A (most polar) through I (least polar). 3-Methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital, Aroclor 1254, pregnenolone-16α-carbonitrile, or ethanol was administered in vivo to genetically “responsive” C57BL/6N or “nonresponsive” DBA/2N mice, in an attempt to understand and identify increases or decreases in reactive BP intermediates that bind to DNA. Rises or falls in these peaks are also noted when liver microsomes from control or 3-methylcholanthrene-treated C57BL/6N or DBA/2N mice were incubated in vitro with [³H]benzo[a]pyrene and microsomal enzyme inhibitors such as α-naphthoflavone, metyrapone or cyclohexene oxide. All of our interpretations concerning the binding of metabolites to DNA are consistent with non-K-region oxygenation of benzo[a]pyrene being mediated predominantly by cytochrome(s) P₁-450 and K-region oxygenation of benzo[a]pyrene being catalysed predominantly by form(s) of P-450 other than P₁-450. All of the biological perturbations are consistent with the following assignments. The major reactive intermediate of benzo[a]pyrene contributing to each peak is suggested to be: peaks A and C, an unknown dihydrodiol oxide; peaks B, D, F and I, quinones oxygenated further (or quinone-derived free radicals); peak E, both cis- and tans-7,8-diol-9,10-epoxides; peak F′, the 7.8-oxide; peak G, the 4.5-oxide; and peak H, an unknown phenol oxide. The DNA nucleosides are not identified in this study. Of the ten peaks listed here, it is of interest that the major metabolite(s) contributing to eight of the peaks (all except peaks F' and G) involve(s) more than a single mono-oxygenation by forms of cytochrome P-450. All peaks, with the exception of peak G, appear to be predominantly associated with benzo[a]pyrene metabolism mediated by P₁-450 and, therefore, controlled by the Ah locus. The use of these microsomal enzyme inducers or inhibitors—combined with the underlying genetic predisposition of the individual, tissue, or cell culture system under study—demonstrates that the balance between P-450 and epoxide hydrase, and the ratio of each form of P-450 to the other forms of P-450, can influence markedly the quantity and quality of reactive intermediates of benzo[a]pyrene that bind to DNA.     

Epigallocatechin-3-gallate does not affect the activity of enzymes involved in metabolic activation and cellular excretion of benzo[a]pyrene in human colon carcinoma cells

Benzo[a]pyrene (B[a]P) and related procarcinogens found in cigarette smoke and roasted foodstuff require metabolic activation to build mutagenic DNA adducts that may cause tumor diseases like colorectal cancer. The major B[a]P-activating enzymes belong to the cytochrome-P450 (CYP)-1 family and are regulated by the aryl hydrocarbon receptor (AhR). Previous studies have indicated that an inhibition of AhR is accompanied with a reduced metabolic activation of B[a]P and therefore may act protective against carcinogenesis. We investigated if the green tea flavonoid (−)-epigallocatechin-3-gallate (EGCG), a known AhR inhibitor, is able to influence B[a]P-metabolizing and B[a]P-transporting enzymes in human Caco-2 colon carcinoma cells. Strikingly, treatment with EGCG did neither affect constitutive and B[a]P-inducible expression of CYP1A1 and UDP-glucuronosyltransferase (UGT)-1A1 nor overall CYP1 and UGT enzyme activities, indicating that EGCG does not antagonize the AhR in Caco-2 cells. Since flavonoids were also identified to enhance the activity of B[a]P-carrying transporter, we analyzed if EGCG exposure alters cellular excretion of B[a]P conjugates. In contrast to the positive control fisetin, EGCG did not affect cellular excretion of B[a]P metabolites. Our data provide evidence that EGCG does not alter the metabolism and transport of B[a]P in Caco-2 cells, and thus may not protect against procarcinogenic food contaminants.     

Comparison of DNA adduct induction in vitro by PAHs and nitro-pahs in freshly isolated rat hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs have been identified widely in occupational and environmental pollution, such as diesel engine emissions and other combustion products. In most cases, hepatic biotransformation is involved in converting these chemicals to their carcinogenic metabolites. It has been demonstrated that isolated hepatocytes possess substantial amounts of the enzymes responsible for metabolizing xenobiotics and are therefore a convenient model for studying chemicals that require activation to exert their carcinogenic effects. In this study, rat hepatocytes were isolated by collagenase digestion and then exposed to benzo[a]pyrene (B) [a]P), benzo[a]anthracene (B[a]A), 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-DNP) at different doses and/or times so that DNA adducts levels, as measured with the ³²P-postlabelling technique, could be compared. Each of the four compounds tested induced significant increases of total DNA adducts with clear dose-related responses. One or more individual adducts were identified as major adducts for each compound. Time-related increases of DNA adducts were also observed from 1 to 4 hr of incubation. Greater amounts of DNA adducts were induced by B[a]P or 1,6-DNP than by B[a]A or 1-NP, with potency being in the order 1,6-DNP > B[a]P > 1-NP B[a]A. These results demonstrate that freshly isolated hepatocytes can be used as an effective in vitro system for the detection of DNA adducts using ³²P-postlabelling, and have shown 1,6-DNP to be the most potent of the tested constituents of diesel emissions.     

Subchronic inhalation of mixtures of cigarette smoke constituents in Xpa-/-p53+/- knock-out mice: a comparison of intermittent with semi-continuous exposure to acetaldehyde, formaldehyde, and acrolein.

We investigated whether inhaling peak concentrations of aldehydes several times daily is more damaging than semi-continuously inhaling low-dose aldehydes. We exposed Xpa-/-p53+/- knock-out mice either intermittently or semi-continuously to mixed acetaldehyde, formaldehyde, and acrolein. The intermittent regimen entailed exposure to the aldehydes 7 min every 45 min, 12 times/day, 5 days/week, corresponding to concentrations inhaled by smokers. Semi-continuously exposed animals received half the dose of aldehydes in 8h/day, 5 days/week. Some mice in each group were sacrificed after 13 weeks of exposure; the rest breathed clean air until the end of 1 year. Mice injected intratracheally with benzo[a]pyrene formed a positive control group. The nasal cavity, lungs, and any macroscopically abnormal organs of all mice were analysed histopathologically. After 13 weeks of exposure, the subacute, overall, histopathological changes induced by the inhalation differed noticeably between the intermittently and semi-continuously treated Xpa-/-p53+/- knock-out mice. After 13 weeks of mixed aldehyde exposure, atrophy of the olfactory epithelium generally appeared, but disappeared after 1 year (adaptation and/or recovery). Respiratory epithelial metaplasia of the olfactory epithelium occurred at a higher incidence at 1 year. Except for a significantly greater number of tumours observed in knock-out mice compared to wild mice (semi-continuous aldehyde exposure and controls), no differences between the semi-continuous and intermittent exposure groups were observed.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)-DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P-DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [³H] B[a]P, but increased the cellular discharge of [³H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20-500 μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P-DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.     

Astaxanthin can alter CYP1A-dependent activities via two different mechanisms: Induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer

Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100 mg Ax/kg body weight/day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme.     

Carotenoids as regulators for inter-species difference in cytochrome P450 1A expression and activity in ungulates and rats

Ungulates (deer, cattle and horses) are reported as animal species which show extreme-accelerated metabolism of CYP1A substrates, such as ethoxyresorufin compared to rats. This study was undertaken to investigate whether accumulation of carotenoids is a possible cause for inter-species difference in CYP1A-dependent activity in this group of animals. The relationship between inter-species differences in CYP1A-dependent activity and the accumulated carotenoids and retinoids as candidates of dietary CYP1A inducers in ungulate species was clarified. Interestingly, there were positive correlations between the accumulated carotenoids, such as β-carotene, with both EROD activity and CYP1A protein expression. These correlations were negative with the accumulated retinoids, such as retinol. The β-carotene was major component of carotenoids in ungulates, and known as an inducer of CYP1A. On the other hand, the retinol is reported as the inhibitor of CYP1A. Other factors which affect CYP1A1 expression, such as polycyclic aromatic hydrocarbons, were also analyzed. To cancel the effects of inter-species difference in CYP1A induction signal cascade among these animals, the rat cell line (H4-II-cells) was treated with the extracted carotenoids from the examined animals. In conclusion, carotenoids and retinoids may have direct effects on the inter-species differences in CYP1A-dependent activity and protein expression.     

Genotoxic activities of the food contaminant 5-hydroxymethylfurfural using different in vitro bioassays

5-Hydroxymethylfurfural (5-HMF) is known as an indicator of quality deterioration in a wide range of foods. 5-HMF is formed as an intermediate in the Maillard reaction and has been identified in a wide variety of heat-processed foods. In recent years, the presence of 5-HMF in foods has raised toxicological concerns: data have shown cytotoxic, genotoxic and tumoral effects but further studies suggest that 5-HMF does not pose a serious health risk. However the subject is still a matter of debate. We investigated the genotoxicity of the food-borne contaminant 5-HMF using the Ames test, the micronucleus (MN) and the single-cell gel electrophoresis (SCGE) assays in the human metabolically active HepG2 cell line. Cytotoxic effect of 5-HMF was first assessed using Alamar Blue™ as a sensitive sub-lethal assay. 5-HMF did not induce any genic mutation in bacteria whatever the concentration in the Ames test. Furthermore, it does not induce clastogenic or aneugenic effects in the HepG2 cells. In contrast, 5-HMF induced HepG2 DNA damage at concentrations from 7.87 to 25 mM in the comet assay suggesting a weak genotoxic effect of 5-HMF in the HepG2 cells probably repaired.     

Assessment of the protective effects of selected dietary anticarcinogens against DNA damage and cytogenetic effects induced by benzo[a]pyrene in C57BL/6J mice

The protective action in C57BL/6 J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen®) as well as chlorophyllin (CHL) against benzo[a]pyrene (B[a]P)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to B[a]P-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30 mg B[a]P/kg b.w. did not differ from those found for animals exposed to B[a]P and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with B[a]P. CHL or EA showed a significant protective effect against B[a]P-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for B[a]P alone.     

Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.     

Safety evaluation of chicken breast extract containing carnosine and anserine.

Chicken breast extract (CBEX) is obtained via hot water extraction of chicken breast and contains among its primary constituents carnosine and anserine, which are histidine-containing dipeptides present in the muscle tissues of most vertebrate species. Dietary intake of CBEX has been previously shown to buffer hydrogen ions formed during high-intensity exercise in human skeletal muscle cells, thereby inhibiting a decrease in muscle cell pH and subsequent muscle fatigue. The objective of this paper is to report the results of safety studies completed on CBEX. CBEX was determined to have an oral LD(50) value of more than 6000 mg/kg body weight in rats. Gavage doses of 500 or 2000 mg CBEX/kg body weight/day administered to rats for 90 days produced no toxicologically significant, dose-related, differences between control and treated animals with respect to body weight gain, food consumption, behavioral effects, hematological and clinical chemistry parameters, absolute and relative organ weights, or gross and microscopic findings. In the presence or absence of metabolic activation, CBEX exerted no mutagenic activity in the Ames assay conducted in various strains of Salmonella typhimurium and Escherichia coli. The results of these studies support the safety of CBEX as a potential dietary source of carnosine and anserine.