Mutagenicity Of Benzo Research Articles

The influence of alkyl substitution on the in vitro metabolism and mutagenicity of benzo[a]pyrene

In recent years concerns over consumer exposure to mineral oil aromatic hydrocarbons (MOAH), especially those containing alkylated polycyclic aromatic hydrocarbons (PAHs), have emerged. This is especially due to the fact that some PAHs are known to be genotoxic and carcinogenic upon metabolic activation. However, available toxicological data on PAHs mainly relate to non-substituted PAHs with limited data on alkyl substituted PAHs. Therefore, the aim of the present study was to characterize in more detail the effect of alkyl substitution on the metabolism and mutagenicity of benzo[a]pyrene (B[a]P), a PAH known to be genotoxic and carcinogenic. To this end, the oxidative metabolism and mutagenicity of B[a]P and a series of its alkyl substituted analogues were quantified using in vitro microsomal incubations and the Ames test. The results obtained reveal that upon alkylation the metabolic oxidation shifts to the aliphatic side chain at the expense of aromatic ring oxidation. The overall metabolism, including metabolism via aromatic ring oxidation resulting potentially in bioactivation, was substantially reduced with elongation of the alkyl side chain, with metabolism of B[a]P with an alkyl substituent of >6 C atoms being seriously hampered. In the Ames test upon metabolic activation, the methyl substitution of B[a]P resulted in an increase or decrease of the mutagenic potency depending on the substitution position. The relevant pathways for mutagenicity of the selected monomethyl substituted B[a]P may involve the formation of a 7,8-dihydrodiol-9,10-epoxide, a 4,5-oxide and/or a benzylic alcohol as an oxidative side chain metabolite which subsequently may give rise to an unstable and reactive sulfate ester conjugate. It is concluded that alkylation of B[a]P does not systematically reduce its mutagenicity in spite of the metabolic shift from aromatic to side chain oxidation.

Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium.

ABSTRACTChromids (secondary chromosomes) in bacterial genomes that are present in addition to the main chromosome appear to be evolutionarily conserved in some specific bacterial groups. In rare cases among these groups, a small number of strains from Rhizobiales and Vibrionales were shown to possess a naturally fused single chromosome that was reported to have been generated through intragenomic homologous recombination between repeated sequences on the chromosome and chromid. Similar examples have never been reported in the family Burkholderiaceae, a well-documented group that conserves chromids. Here, an in-depth genomic characterization was performed on a Burkholderiaceae bacterium that was isolated from a soil bacterial consortium maintained on diesel fuel and mutagenic benzo[a]pyrene. This organism, Cupriavidus necator strain KK10, was revealed to carry a single chromosome with unexpectedly large size (>6.6 Mb), and results of comparative genomics with the genome of C. necator N-1T indicated that the single chromosome of KK10 was generated through fusion of the prototypical chromosome and chromid at the rRNA operons. This fusion hypothetically occurred through homologous recombination with a crossover between repeated rRNA operons on the chromosome and chromid. Some metabolic functions that were likely expressed from genes on the prototypical chromid region were indicated to be retained. If this phenomenon—the bacterial chromosome-chromid fusion across the rRNA operons through homologous recombination—occurs universally in prokaryotes, the multiple rRNA operons in bacterial genomes may not only contribute to the robustness of ribosome function, but also provide more opportunities for genomic rearrangements through frequent recombination.IMPORTANCE A bacterial chromosome that was naturally fused with the secondary chromosome, or “chromid,” and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae. Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family. This fusion has hypothetically occurred through intragenomic homologous recombination between repeated rRNA operons and, if so, provides novel insight into the potential of multiple rRNA operons in bacterial genomes to lead to chromosome-chromid fusion. The harsh conditions under which strain KK10 was maintained—a genotoxic hydrocarbon-enriched milieu—may have provided this genotype with a niche in which to survive.

Detoxification of Oral Exposure to Benzo(a)pyrene by Lactobacillus plantarum CICC 23121 in Mice.

This study's previous work showed that the carcinogen and mutagen benzo(a)pyrene (BaP) can be adsorbed by Lactobacillus cells in vitro. However, in vivo BaP detoxification by lactic acid bacteria has not yet been investigated. The present study evaluates the effects of orally administered Lactobacillus plantarum CICC 23121 in BaP-treated mice. Oral administration of 50mg kg-1 BaP perturbed the intestinal microflora, caused Proteobacteria to predominate, and severely damaged DNA. However, oral administration of 5 × 1010 CFU mL-1 CICC 23121 in BaP-treated mice enhances fecal BaP excretion from 181.70± 1.04µg/(g∙h) to 271.47± 11.71µg/(g∙h) after 6 h. Fecal BaP excretion reaches up to 280.66± 22.97µg/(g∙h) after the first 4 days of orally administered CICC 23121 and decreased to 94.31± 2.64µg/(g∙h) by day 11. Intestinal microbiota are restored and Firmicutes predominates. CICC 23121 alleviates BaP-induced DNA damage and reduces tail length from 56.37± 5.31 to 39.69± 4.27µm. Therefore, oral CICC23121 consumption is a promising strategy for reducing BaP toxicity in mice. To the best of our knowledge, this report is the first report to demonstrate in vivo that Lactobacillus cells can detoxify BaP.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were −2.5–3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 β−hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

Integrated in silico and in vitro genotoxicity assessment of thirteen data-poor substances

The Canadian Domestic Substances List (DSL) contains chemicals that have not been tested for genotoxicity as their use pre-dates regulatory requirements. In the present study, (quantitative) structure-activity relationships ((Q)SAR) model predictions and in vitro tests were conducted for genotoxicity assessment of 13 data-poor chemicals from the DSL (i.e. CAS numbers 19286-75-0, 13676-91-0, 2478-20-8, 6408-20-8, 74499-36-8, 26694-69-9, 29036-02-0, 120-24-1, 84696-48-9, 4051-63-2, 5718-26-3, 632-51-9, and 600-14-6). First, chemicals were screened by (Q)SAR models in Leadscope® and OASIS TIMES; two chemicals were excluded from (Q)SAR as they are complex mixtures. Six were flagged by (Q)SAR as potentially mutagenic and were subsequently confirmed as mutagens using the Ames assay. Of nine chemicals with clastogenic (Q)SAR flags, eight induced micronuclei in TK6 cells. Benchmark dose analysis was used to evaluate the potency of the chemicals. Four chemicals were bacterial mutagens with similar potencies. Three chemicals were more potent in micronuclei induction than the prototype alkylating agent methyl methanesulfonate and three were equipotent to the mutagenic carcinogen benzo[a]pyrene in the presence of rat liver S9. Overall, 11 of the 13 DSL chemicals demonstrated at least one type of genotoxicity in vitro. This study demonstrates the application of genotoxic potency analysis for prioritizing further investigations.

Effect of interleukin (IL)-8 on benzo[a]pyrene metabolism and DNA damage in human lung epithelial cells.

It has been well established that inflammation and concurrent mutagenic exposures drive the carcinogenic process in a synergistic way. To elucidate the role of the inflammatory cytokine IL-8 in this process, we studied its effect on the activation and deactivation of the chemical mutagen benzo[a]pyrene B[a]P in the immortalized pulmonary BEAS-2B cell line. After 24h incubation with B[a]P in the presence or absence of IL-8, the B[a]P induced cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) gene expression and CYP1A1 enzyme activity was significantly higher in the presence of the cytokine. Consistent with these findings, we observed higher concentration of the metabolite B[a]P-7,8-diol under concurrent IL-8 treatment conditions. Interestingly, we also found higher concentrations of unmetabolized B[a]P. To explain this, we examined the downstream effects of IL-8 on NADPH oxidases (NOXes). IL-8 lowered the intracellular NADPH level, but this effect could not explain the changes in B[a]P metabolism. IL-8 also significantly depleted intracellular glutathione (GSH), which also resulted in enhanced levels of unmetabolized B[a]P, but increased concentrations of the metabolite B[a]P-7,8-diol. No differences in B[a]P-DNA adducts level were found between B[a]P and B[a]P combined with IL-8, and this might be due to a 3-fold increase in nucleotide excision repair (NER) after IL-8 treatment. These findings suggest that IL-8 increased the formation of B[a]P-7,8-diol despite an overall delayed B[a]P metabolism via depletion of GSH, but DNA damage levels were unaffected due to an increase in NER capacity.

Oxidative Stress Levels in Buccal Cells Using MAWI Collection Tubes

Background: Oxidative Stress (OS) is defined as an imbalance between oxidants and antioxidants, in favor of oxidants, potentially leading to DNA damage. Benzo[a] pyrene (B(a)P), a representative DNA-damaging mutagenic/ carcinogenic Polycyclic Aromatic Hydrocarbons (PAH), can lead to the final mutagen Benzo(a)Pyrene Diol Epoxide (BPDE). Methods: The extent of oxidative DNA damage is investigated in population studies using easily obtained cells. Buccal cell usage has been shown by many to be a cost effective, non-invasive and safe method to isolate DNA for various biological experiments. In this experimental research of 40 participants, equally divided between industry and academia, we compared the DNA concentration, purity, and associated levels of BPDE-DNA damage. Buccal cells were collected using ISWAB-DNA tubes, and DNA was then extracted to study the extent of DNA damage via ELISA Kit. Results: Results showed pure samples with no DNA degradation. DNA yields were as high as 35.657 μg/mL. In addition, none of the samples showed a presence of BPDE-DNA damage. Conclusions: MAWI collection tubes may not be able to detect BPDE-DNA damage. Other OS markers should be used to eradicate the previous statement.

In Utero Exposure to Benzo[a]Pyrene Increases Mutation Burden in the Soma and Sperm of Adult Mice.

Background:Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear.Objectives:We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) has an impact on the burden and distribution of mutations in adult mice.Methods:We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development.Results:Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of first filial generation (F1) males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on the reproductive function of F1 males.Conclusion:In utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently affecting both the genetic health of the F1 and the population gene pool.Citation:Meier MJ, O’Brien JM, Beal MA, Allan B, Yauk CL, Marchetti F. 2017. In utero exposure to benzo[a]pyrene increases mutation burden in the soma and sperm of adult mice. Environ Health Perspect 125:82–88; http://dx.doi.org/10.1289/EHP211

Characterizing Benzo[a]pyrene-induced lacZ mutation spectrum in transgenic mice using next-generation sequencing.

BackgroundThe transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening.MethodsWe developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS.ResultsWe identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C → T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %.ConclusionThese results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2004-4) contains supplementary material, which is available to authorized users.

Effects of Antrodia camphorata extracts on anti-oxidation, anti-mutagenesis and protection of DNA against hydroxyl radical damage.

BackgroundAntrodia camphorata is a geographically special fungus and is one of the precious traditional medicines of Taiwan. A lot of reports have addressed its antioxidant activities and anticancer activities. In order to understand whether these protection effects were resulted from its ability of preventing DNA against hydroxyl radical damage, the A. camphorata extract was used to examine its antioxidant, antimutagenic and DNA-protective activities.MethodsA. camphorata extract was prepared by extracting the lyophilized powder of A. camphorata mycelium with distilled water. The antioxidative activity of this A. camphorata extract was then evaluated by 2,2-diphenyl-1-picrylhydrozyl (DPPH) radical-scavenging assay, and the antimutagenic activities of the extract against direct mutagen 4-nitroquinoline N-oxide (4NQNO) and indirect mutagen benzo[a]pyrene (B[a]P) were evaluated by Ames test. The effects of the A. camphorata extract in terms of DNA protection against hydroxyl radical damage were also investigated.ResultsIt was found that the higher the concentration of A. camphorata extracts, the higher the DPPH radical-scavenging effect. A. camphorata extract at concentrations between 0.625 and 10 mg/ml was found to be neither toxic nor mutagenic. However, the higher A. camphorata concentration (10 mg/ml) used in the test showed higher inhibitory effects on 4NQNO in a dose-dependent manner. The A. camphorata extract also showed reducing and scavenging activities against superoxide anion radical and also exhibited protective effects on DNA against hydroxyl radical-induced damage.ConclusionsResults suggested that A. camphorata is a non-toxic and novel material with antioxidant, antimutagenic and DNA-protective activities and could be developed into health foods.

Hypoxia diminishes the detoxification of the environmental mutagen benzo[a]pyrene.

Hypoxia promotes genetic instability and is therefore an important factor in carcinogenesis. We have previously shown that activation of the hypoxia responsive transcription factor HIFα can enhance the mutagenic phenotype induced by the environmental mutagen benzo[a]pyrene (BaP). To further elucidate the mechanism behind the ability of hypoxia to increase mutagenicity of carcinogens, we examined the activation and detoxification of BaP under hypoxic conditions. To this end, the human lung carcinoma cell line A549 was treated with BaP under 20%, 5% or 0.2% oxygen for 18h and alterations in BaP metabolism were assayed. First, BaP-induced expression of key metabolic enzymes was analysed; expression levels of the activating CYP1A1 and CYP1B1 were increased, while the detoxifying enzymes UGT1A6 and UGT2B7 were significantly reduced by hypoxia. To evaluate whether these changes had an effect on metabolism, levels of BaP and several of its metabolites were determined. Cells under hypoxia have a reduced capacity to metabolise BaP leaving more of the parent molecule intact. Additionally, BaP-7,8-dihydrodiol, the pre-cursor metabolite of the reactive metabolite BaP-7,8-dihydroxy-9,10-epoxide (BPDE), was formed in higher concentrations. Finally, under hypoxia, DNA adducts accumulated over a period of 168 h, whereas adducts were efficiently removed in 20% oxygen conditions. The delayed detoxification kinetics resulted in a 1.5-fold increase in DNA adducts. These data indicate that the metabolism under hypoxic conditions has shifted towards increased activation of BaP instead of detoxification and support the idea that modulation of carcinogen metabolism is an important additional mechanism for the observed HIF1 mediated genetic instability.

Antimutagenic and anticancer activity of Darjeeling tea in multiple test systems.

BackgroundDarjeeling tea, a most popular variety of black tea, though consumed by the people in different parts of world but its beneficial health effects have not been investigated in details. In this study, the antimutagenic and anticancer effect of Darjeeling tea extract (DTE) has been evaluated.MethodsAntimutagenic activity of the DTE was carried out in two different strains of Salmonella typhimurium by AMES test against a known mutagen benzo[a]pyrene (B[a]P) with S9 activation. Moreover, anticlastogenic property of DTE was also measured by micronuclei formation (MN) against B[a]P with S9 activation in human lymphocytes. The anticancer activity of the same was studied on U937 cell line. Here, Human PBMCs were used as the normal cell control to identify selective anticancer activity of the extract against U937 cells.ResultsThe results showed significant antimutagenic activity on bacterial strains. A significant decrease in MN was also observed in the DTE treated human lymphocyte cultures pretreated with B[a]P when compared with B[a]P treated cultures alone. The study clearly exhibited anticancer activity of the extract on U937 cell line. Further studies also revealed that apoptosis induction is an important mechanism behind the anticancer effect of DTE.ConclusionOverall, this study indicates that DTE has significant antimutagenic and anticancer activities on bacterial and mammalian cells respectively.

Early-life exposure to benzo[a]pyrene increases mutant frequency in spermatogenic cells in adulthood.

Children are vulnerable to environmental mutagens, and the developing germline could also be affected. However, little is known about whether exposure to environmental mutagens in childhood will result in increased germline mutations in subsequent adult life. In the present study, male transgenic lacI mice at different ages (7, 25 and 60 days old) were treated with a known environmental mutagen (benzo[a]pyrene, B[a]P) at different doses (0, 50, 200 or 300 mg/kg body weight). Mutant frequency was then determined in a meiotic cell type (pachytene spermatocyte), a post-meiotic cell type (round spermatid) and epididymal spermatozoa after at least one cycle of spermatogenesis. Our results show that 1) mice treated with B[a]P at 7 or 25 days old, both being pre-adult ages, had significantly increased mutant frequencies in all spermatogenic cell types tested when they were 60 days old; 2) spermatogenic cells from mice treated before puberty were more susceptible to B[a]P-associated mutagenesis compared to adult mice; and 3) unexpectedly, epididymal spermatozoa had the highest mutant frequency among the spermatogenic cell types tested. These data show that pre-adult exposure to B[a]P increases the male germline mutant frequency in young adulthood. The data demonstrate that exposure to environmental genotoxins at different life phases (e.g., pre-adult and adult) can have differential effects on reproductive health.

POTENTIAL ANTICLASTOGENIC EFFECT OF HYPERFORIN

Summary Hyperforin is a prenylated phloroglucinol derivative which occurs in the plant Hypericum perforatum L. It has several medicinally important properties (antidepressant, anti-inflammatory, proapoptotic, antibacterial and antiangiogenic). To enable its medicinal use, it is necessary to investigate its potential genotoxic effect on human cell lines. We also observed an anticlastogenic effect of hyperforin towards the indirect mutagen benzo(a)pyrene. Benzo(a)pyrene is a widespread polycyclic aromatic hydrocarbon. We performed experiments on human tumor cell line HepG2, using the in vitro mammalian chromosome aberration test. We investigated two cytogenic parameters: the number of aberrant metaphases and the total number of chromosome aberrations. We found out that hyperforin was not genotoxic on human cell line HepG2. The second part of our results implies that hyperforin had an anticlastogenic effect against the indirect mutagen benzo(a)pyrene in our experimental conditions. In future we will continue our research by using another range of hyperforin concentrations, other cell lines and other chemical mutagens.

Role of Structural and Energetic Factors in Regulating Repair of a Bulky DNA Lesion with Different Opposite Partner Bases

Extensive molecular modeling with molecular dynamics simulations and van der Waals energy analyses were used to elucidate the striking finding that a mutagenic benzo[a]pyrene-derived DNA lesion, the base-displaced intercalated 10R-(+)-cis-anti-B[a]P-N(2)-dG (G*), manifests large differences in nucleotide excision repair (NER) efficiencies in DNA duplexes, which depend on the identities of the partner base opposite G*. The nature of the partner base causes marked differences in the extent of its major groove extrusion and dynamics, as well as energetic stability of the intercalation pocket that parallels the relative NER efficiencies.

Abstract 4118: Hypoxia affects the metabolic activation and detoxification of the environmental mutagen benzo(a)pyrene

Abstract Hypoxia is characteristic for a number of pathologies and promotes genetic instability, as exposure to low oxygen levels results in an elevated mutation frequency. We have previously shown that activation of the hypoxia responsive transcription factor HIF-1α, in the presence of sufficient oxygen, enhanced mutation frequencies induced by the environmental mutagen benzo[a]pyrene (BaP). Since proper carcinogen metabolism depends on oxygen we examined how varying oxygen levels influenced BaP metabolism by studying its activation and deactivation. The human lung carcinoma cell line A549 was treated with BaP under different oxygen concentrations (20%, 5%, and 0.2%) for 18 hours and alterations in BaP-metabolism were determined. First, we analyzed the BaP induced expression of key metabolic enzymes; expression levels of CYP1A1 and CYP1B1 was higher under hypoxia, while expression of the detoxifying enzymes UGT1A6 and UGT2B7 were significantly reduced by hypoxia. Then, several BaP metabolites in supernatants were determined by HPLC-fluorescence detection. In general, low oxygen levels lead to slower metabolism of BaP leaving more of the parent molecule intact. At the same time, BaP-7,8-dihydrodiol, the pre-cursor metabolite of the reactive metabolite BaP-7,8-dihydroxy-9,10-epoxide (BPDE), was formed in higher concentrations under hypoxia. Finally, under hypoxia DNA adducts accumulated over a period of 168 hours, whereas DNA adducts were efficiently removed under 20% oxygen, eventually leading to 1.6 times higher adduct levels under hypoxic conditions. These data indicate that the metabolism has shifted towards increased activation of BaP instead of detoxification under hypoxic conditions. These data support the idea that modulation of carcinogen metabolism may be an important additional mechanism for the observed HIF1 mediated genetic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4118. doi:1538-7445.AM2012-4118

Classification of polycyclic aromatic hydrocarbons based on mutagenicity in lung tissue through DNA microarray

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. Exposure to PAHs raises the risk of lung cancer and inflammatory and allergic disorders such as asthma. DNA microarray technologies have been applied to research on toxicogenomics in the recent years. To evaluate the mutagenicity of PAHs and constituents of environmental pollutants in lung tissue, including metabolic activation, human alveolar epithelial type II cells (A549) were treated with nonmutagenic PAH pyrene and with the mutagenic PAHs benzo-[a]-pyrene, 1-nitropyrene, or 1,8-dinitropyrene. Comparison of genome-wide microarray expression profiles between a nonmutagenic and a mutagenic PAH-treated group revealed that xenobiotic response genes such as CYP1B1 were commonly upregulated in two groups and that DNA damage induced genes, especially p53-downstream genes such as p21 (CDKN1A) were upregulated only in the mutagenic PAH-treated group. Pretreatment with cytochrome P450 inhibitor α-naphthoflavone or p53 inhibitor pifithrin-α inhibited the benzo-[a]-pyrene-induced p21 expression. These data suggest that when PAHs enter the cells, lung epithelium induces PAH metabolic activating enzymes, and then the DNA damages-recognition signal is converged with p53 downstream genes. This metabolic activation and DNA damage is induced in lung epithelium, and the mutagenicity of PAHs can be classified by DNA microarray expression profiles.

Pulmonary gene and microRNA expression changes in mice exposed to benzo(a)pyrene by oral gavage

Exposure to the environmental mutagen benzo(a)pyrene (BaP) alters the expression of AHR-responsive genes as well as genes involved in other pathways. We recently reported that exposure of adult mice to BaP resulted in a robust transcriptome response in the liver, but this was accompanied by a complete lack of change in microRNA (miRNA) expression. Since BaP exposure does not result in hepatocarcinogenicity, but does cause lung cancer, in the present study we examine the pulmonary mRNA and miRNA responses to BaP in the same mice. Adult male B6C3F1 mice were exposed to 150 and 300mg/kg BaP by oral gavage for three consecutive days and sacrificed 4h after the last exposure. Serum clinical chemistry was performed for both the doses to assess the general toxicity of BaP; a modest decrease in serum inorganic phosphorous was observed at both the doses. A small decrease in serum glucose following 150mg/kg and alkaline phosphatase following 300mg/kg BaP was observed. BaP–DNA adduct levels in whole lung and liver tissues were assessed by 32P postlabelling and similar dose dependent increases were observed for lung and liver. Using DNA microarrays, pulmonary mRNA and miRNA expressions were analysed. Over 1000 genes were statistically differentially expressed (p<0.05). The perturbed pathways included oxidative stress, xenobiotic metabolism, cell proliferation, cell cycle, B and T-cell receptor signalling and primary immunodeficiency signalling pathways. Analysis of miRNA profiles revealed downregulation of miR-150, miR-142-5p, miR-122 and upregulation of miR-34c, miR-34b-5p and miR-29b. These miRNAs are involved in the biological processes, immune response, cell proliferation and cell cycle, which are the main pathways affected at the mRNA level. Thus, miRNAs are more responsive to BaP in lungs than in liver, and are likely to be involved in the regulation of the pulmonary responses to BaP exposure.

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 (100mg Ax/kg body weight/day for 3days) 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.

Prospective analysis of DNA damage and repair markers of lung cancer risk from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial

Mutagen challenge and DNA repair assays have been used in case-control studies for nearly three decades to assess human cancer risk. The findings still engender controversy because blood was drawn after cancer diagnosis so the results may be biased, a type called 'reverse causation'. We therefore used Epstein-Barr virus-transformed lymphoblastoid cell lines established from prospectively collected peripheral blood samples to evaluate lung cancer risk in relation to three DNA repair assays: alkaline Comet assay, host cell reactivation (HCR) assay with the mutagen benzo[a]pyrene diol epoxide and the bleomycin mutagen sensitivity assay. Cases (n = 117) were diagnosed with lung cancer between 0.3 and 6 years after blood collection and controls (n = 117) were frequency matched on calendar year and age at blood collection, gender and smoking history; all races were included. Case and control status was unknown to laboratory investigators. In unconditional logistic regression analyses, statistically significantly increased lung cancer odds ratios (OR(adjusted)) were observed for bleomycin mutagen sensitivity as quartiles of chromatid breaks/cell [relative to the lowest quartile, OR = 1.2, 95% confidence interval (CI): 0.5-2.5; OR = 1.4, 95% CI: 0.7-3.1; OR = 2.1, 95% CI: 1.0-4.4, respectively, P(trend) = 0.04]. The magnitude of the association between the bleomycin assay and lung cancer risk was modest compared with those reported in previous lung cancer studies but was strengthened when we included only incident cases diagnosed more than a year after blood collection (P(trend) = 0.02), supporting the notion the assay may be a measure of cancer susceptibility. The Comet and HCR assays were unrelated to lung cancer risk.