Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously distributed environmental chemicals. PAHs acquire carcinogenicity only after they have been activated by xenobiotic-metabolizing enzymes to highly reactive metabolites capable of attacking cellular DNA. Cytochrome P450 (CYP) enzymes are central to the metabolic activation of these PAHs to epoxide intermediates, which are converted with the aid of epoxide hydrolase to the ultimate carcinogens, diol-epoxides. Historically, CYP1A1 was believed to be the only enzyme that catalyzes activation of these procarcinogenic PAHs. However, recent studies have established that CYP1B1, a newly identified member of the CYP1 family, plays a very important role in the metabolic activation of PAHs. In CYP1B1 gene-knockout mice treated with 7,12-dimethyl-benz[a]anthracene and dibenzo[ a, l ]pyrene, decreased rates of tumor formation were observed, when compared to wild-type mice. Significantly, gene expression of CYP1A1 and 1B1 is induced by PAHs and polyhalogenated hydrocarbons such as 2,3,7,8-tetrachlorodibenzo- p -dioxin through the arylhydrocarbon receptor. Differences in the susceptibility of individuals to the adverse action of PAHs may, in part, be due to differences in the levels of expression of CYP1A1 and 1B1 and to genetic variations in the CYP1A1 and 1B1 genes.     

Modulation of gene expression and DNA adduct formation in HepG2 cells by polycyclic aromatic hydrocarbons with different carcinogenic potencies

Polycyclic aromatic hydrocarbons (PAHs) can occur in relatively high concentrations in the air, and many PAHs are known or suspected carcinogens. In order to better understand differences in carcinogenic potency between PAHs, we investigated modulation of gene expression in human HepG2 cells after 6 h incubation with varying doses of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), fluoranthene (FA), dibenzo[a,h]anthracene (DB[a,h]A), 1-methylphenanthrene (1-MPA) or dibenzo[a,l]pyrene (DB[a,l]P), by using cDNA microarrays containing 600 toxicologically relevant genes. Furthermore, DNA adduct levels induced by the compounds were assessed with (32)P-post-labeling, and carcinogenic potency was determined by literature study. All tested PAHs, except 1-MPA, induced gene expression changes in HepG2 cells, although generally no dose-response relationship could be detected. Clustering and principal component analysis showed that gene expression changes were compound specific, since for each compound all concentrations grouped together. Furthermore, it showed that the six PAHs can be divided into three groups, first FA and 1-MPA, second B[a]P, B[b]F and DB[a,h]A, and third DB[a,l]P. This grouping corresponds with the carcinogenic potencies of the individual compounds. Many of the modulated genes are involved in biological pathways like apoptosis, cholesterol biosynthesis and fatty acid synthesis. The order of DNA adduct levels induced by the PAHs was: B[a]P > DB[a,l]P > B[b]F > DB[a,h]A > 1-MPA >/= FA. When comparing the expression change of individual genes with DNA adduct levels, carcinogenic potency or Ah-receptor antagonicity (the last two were taken from literature), several highly correlated genes were found, of which CYP1A1, PRKCA, SLC22A3, NFKB1A, CYP1A2 and CYP2D6 correlated with all parameters. Our data indicate that discrimination of high and low carcinogenic PAHs by gene expression profiling is feasible. Also, the carcinogenic PAHs induce several pathways that were not affected by the least carcinogenic PAHs.     

CYP1A1 Inducing Potential of Airborne Particulate Extracts Collected during a 25-year Period (1975-2000)

Samples of airborne particles from Sapporo, the capital of Hokkaido, Japan, were collected between 1975 and ‍2000. Major polycyclic aromatic hydrocarbons (PAHs) included in the extracts of airborne particles were investigated ‍for their mutagenicity and potential for inducing drug-metabolizing enzyme cytochrome P450 (CYP)1A1, which is ‍considered to be responsible for the activation of PAHs in airborne particle extracts, as well as in cigarette smoke, to ‍carcinogens and is associated with risk of several cancers. There was a dose-related increase in CYP1A1 activity in ‍human lymphoblastoid cells after exposure to airborne particulates containing PAHs. The mutagenicity of the ‍airborne particles collected in summer was lowest and for those collected in spring was lower than in autumn or ‍winter. Likewise, the winter sample had the strongest CYP1A1 inducing potential while the summer sample had the ‍weakest. CYP1A1 inducing potency was strongly related to the amount of benzo(k)fluorathene (Spearman’s rank ‍correlation coefficient (ã) = 0.97), benzo[a]pyrene ã = 0.96), benzo[g,h,i]perylene (ã = 0.94), benz[a]anthracene (ã = ‍0.93), chrysene (ã = 0.93) in the extracts during the 25-year period, while the enzyme activity was measurably related ‍to the amount of pyrene (ã = 0.64) and fluorathene (ã = 0.54). During the 25-year period, CYP1A1 inducing potential ‍decreased every year together with a decrease in PAHs in the airborne particle extracts. CYP1A1 inducing potential ‍may be one of the most convenient biomarkers with which to estimate the overall carcinogenicity/mutagenicity of ‍airborne particle extracts. ‍     

Dibenzo[A,L]pyrene-induced genotoxic and carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice

Dibenzo[a,l]pyrene (DB[a,l]P), a notorious air pollutant, is the most powerful carcinogenic polycyclic aromatic hydrocarbon (PAH) ever tested. Although the carcinogenicity of PAH may be primarily mediated by the aryl hydrocarbon receptor (AhR), the in vivo role of AhR in skin carcinogenesis remains to be defined. In this context, we investigated the genotoxic and carcinogenic responses of the AhR-deficient mouse skin to DB[a,l]P. A single painting resulted in a striking epidermal hyperplasia in AhR+/+ mice but not in AhR-/- mice. Bromodeoxyuridine-labeling index and accumulation of p53 protein in epidermal cells of AhR+/+ mice were 8- and 33-fold higher than those of AhR-/- mice, respectively. 32P-Postlabeling assay for DB[a,l]P-DNA adducts displayed a 2-fold increase in the AhR+/+ mouse skin. After DB[a,l]P exposure, AhR-/- mice arranged a nearly 60% reduction in the induction of epidermal cytochrome P450 (CYP)1A1, but CYP1B1 was constitutively expressed in both genotypes of mice, irrespective of DB[a,l]P treatment. As compared with AhR+/+ mice, AhR-/- mice had both significantly lower incidence (100% vs. 33%) and multiplicity (2.7 vs. 0.46) of skin tumors by the complete carcinogenesis study. These observations indicate that a reduced tumor yield in AhR-/- mice may be secondary to reduction of inducible CYP1A1 activation and subsequent DNA adduction. It is evident from our continuous work that although AhR is likely to play a central role in epidermal proliferation and possibly neoplastic transformation, the relative importance of AhR for carcinogenesis may be different among PAH examined.     

Aryl hydrocarbon receptor signaling plays a significant role in mediating benzo[a]pyrene- and cigarette smoke condensate-induced cytogenetic damage in vivo

This laboratory has previously reported data suggesting that aryl hydrocarbon receptor (AhR) signaling may have a net potentiating effect on the DNA damaging potential of cigarette smoke. The experiments described in this report extend these studies by testing whether the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) can modify the in vivo genetic toxicity of benzo[a]pyrene (B[a]P) and the complex mixture of chemicals in cigarette smoke condensate (CSC). Initial experiments were designed to determine 3'M4'NF doses which can antagonize AhR in vivo but which have little effect on constitutive cytochrome P4501A (CYP1A) activity. These experiments took three forms: (i) zoxazolamine paralysis tests, a functional assay of cytochrome P450 CYP1A activity in 3'M4'NF-treated C57Bl/6J mice; (ii) co-treatment of AHR: null allele mice with 150 mg/kg B[a]P plus a range of 3'M4'NF concentrations in order to evaluate the potential of the flavone to interact with non-AhR targets which may affect B[a]P toxicity; (iii) an evaluation of the in vivo AhR antagonist activity of 3'M4'NF using transgenic mice which carry a dioxin-responsive element-regulated lacZ reporter. Once an appropriate dose range was determined, C57Bl/6J mice were challenged with B[a]P or CSC with and without 3'M4'NF co-treatment. Chromosome damage was measured by scoring the frequency of micronuclei in peripheral blood reticulocytes. Data presented herein suggest that 3'M4'NF can protect mice from B[a]P-induced bone marrow cytotoxicity and genotoxicity. Furthermore, CSC-associated genotoxicity was abolished by the flavonoid. These data add support to our hypothesis that AhR signaling has a net potentiating effect on the genetic toxicity and, presumably, carcinogenicity of cigarette smoke.     

Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagens and stimulators of DNA repair synthesis in cultured human cells

Ten polycyclic aromatic hydrocarbons (PAHs), viz. anthracenepyrene, chrysene, perylene, fluoranthene, benzo[e]pyrene, benzo[a]pyrene,benz[a]anthracene, benzo[ghi]perylene, benzo[k]fluoranthene,have been nitrated using concentrated nitric acid and the crudenitrated mixture examined for biological activity. All the nitroPAHs examined were mutagenic to Salmonella typhimurium in theabsence of a rat liver preparation. Addition of Aroclor-1254induced liver had little effect on mutagenicity. Mutagenic potencydiffered for the various nitrated mixtures with nitrated pyreneand nitrated fluoranthene the most potent and nitrated anthracenethe least potent. Both frame-shift and base-substitution mutationswere induced by the nitrated PAHs. The nitrated PAHs were alsoable to induce DNA repair synthesis in cultured HeLa cells inthe absence of liver, indicating that these cells have the necessaryenzymes to activate nitro PAHs. Potency again varied from compoundto compound with nitrated pyrene appearing to be the most active.Isolation of individual components from the crude nitrated mixtureshas not been carried out in this study. In view of the possiblewide-spread distribution of nitrated PAHs in the environmentfurther work is required to assess the carcinogenic potencyof these compounds which possibly pose a risk to man.     

Oncogenic interaction of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons in mice.

To evaluate possible interactions between PAH occurring in automobile exhaust condensates with regard to their tumour forming potency, the following experiments were performed. Six different doses of benzo[a]pyrene (3-100 microgram) and of dibenzo]a,h]anthracene (2-75 microgram) and mixtures thereof were tested subcutaneously on female NMRI mice. In addition, mixtures of 10 non-carcinogenic hydrocarbons were applied: benzo[e]pyrene, benzo[a]anthracene, phenanthrene, anthracene, pyrene, fluoranthene, chrysene, perylene, benzo[ghi]perylene and coronene. Mixtures of all 12 PAH were also applied. The proportion of PAH in all mixtures used was the same as in automobile exhaust condensates; benzo[a]pyrene was used as reference substance. The most important results were as follows: 1. Small doses of dibenzo[a,h]anthracene have a greater tumour promoting effect than do comparable doses of benzo[a]pyrene. Increased doses increase the effect of benzo[a]pyrene more than that of dibenzo[a,h]anthracene. 2. The mixture of benzo[a]pyrene and dibenzo[a,h]anthracene is 1.4 time more active than dibenzo[a,h]anthracene alone. 3. The mixture of all PAH has a lower efficacy than dibenzo[a,h]anthracene alone, amounting to only 0.03 that of dibenzanthracene; however, the activity of dibenzo[a,h]anthracene within the mixture of the 12 PAH increases by a factor of 3.1. 4. The activity of a mixture of dibenzo[a,h]anthracene and benzo[a]pyrene depends to about 40% on dibenzo[a,h]anthracene; and that of 12 PAH to 30% on dibenzo[a,h]anthracene alone or to 80% on a mixture of dibenzo[a,h]anthracene and benzo[a]pyrene.     

Binary PAH mixtures cause additive or antagonistic effects on gene expression but synergistic effects on DNA adduct formation

Polycyclic aromatic hydrocarbons (PAHs) cover a wide range of structurally related compounds which differ greatly in their carcinogenic potency. PAH exposure usually occurs through mixtures rather than individual compounds. Therefore, we assessed whether the effects of binary PAH mixtures on gene expression, DNA adduct formation, apoptosis and cell cycle are additive compared with the effects of the individual compounds in human hepatoma cells (HepG2). Equimolar and equitoxic mixtures of benzo[a]pyrene (B[a]P) with either dibenzo[a,l]pyrene (DB[a,l]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[b]fluoranthene (B[b]F), fluoranthene (FA) or 1-methylphenanthrene (1-MPA) were studied. DB[a,l]P, B[a]P, DB[a,h]A and B[b]F dose-dependently increased apoptosis and blocked cells cycle in S-phase. PAH mixtures showed an additive effect on apoptosis and on cell cycle blockage. DNA adduct formation in mixtures was higher than expected based on the individual compounds, indicating a synergistic effect of PAH mixtures. Equimolar mixtures of B[a]P and DB[a,l]P (0.1, 0.3 and 1.0 microM) were assessed for their effects on gene expression. Only at 1.0 microM, the mixture showed antagonism. All five compounds were also tested as a binary mixture with B[a]P in equitoxic concentrations. The combinations of B[a]P with B[b]F, DB[a,h]A or FA showed additivity, whereas B[a]P with DB[a,l]P or 1-MPA showed antagonism. Many individual genes showed additivity in mixtures, but some genes showed mostly antagonism or synergism. Our results show that the effects of binary mixtures of PAHs on gene expression are generally additive or slightly antagonistic, suggesting no effect or decreased carcinogenic potency, whereas the effects on DNA adduct formation show synergism, which rather indicates increased carcinogenic potency.     

利用Tox21通路数据库评估石家庄市大气细颗粒物中多环芳烃激活毒性通路的风险

目的： 采用Tox21毒性检测数据库中通路数据对2017年石家庄市供暖期大气细颗粒物（PM_2.5）中多环芳烃（PAHs）的检测数据进行分析计算，评估PM_2.5引起毒性通路激活的风险。方法： 在石家庄市河北医科大学远离工业污染源设置1个采样点收集大气PM_2.5，通过气相色谱-串联质谱法（GC-MS）检测美国环境保护署优先控制的16种PAHs的浓度。利用MPPD软件计算各PAHs单体在成人肺泡中的沉积量；检索Tox21毒性检测数据库中芳烃受体（AhR）、核因子E2相关因子（Nrf2）、p53和核因子κB（NF-κB）各通路的剂量反应关系数据，计算各PAHs单体激活各通路的单位强度；结合PAHs在肺泡的沉积量和PAHs激活通路的强度，评估PM_2.5激活各个通路的风险。结果： 石家庄市供暖期PM_2.5中PAHs检测结果显示，苯并[a]芘的日均浓度为9.13 ng/m³，日均浓度排名前3的苯并[b]荧蒽、荧蒽和芘的浓度分别为22.88、17.86及14.31 ng/m³。这16种PAHs以苯并[a]芘为参照的毒性当量浓度为17.74 ng/m³。毒性通路激活风险预测结果提示激活强度最大的是NF-κB通路，其次是AhR、Nrf2，p53被激活的可能性最弱，其中相对应的活性暴露比（AER）值分别为1.97、1.71、0.58和0.28。结论： 石家庄市大气细颗粒物中PAHs的暴露，可能通过激活NF-κB和AhR信号通路增加致癌风险。     

Inhibitory effect of Phenethyl Isothiocyanate Against Benzo[a] Pyrene-Induced Rise in CYP1A1 mRNA and Apoprotein Levels as its Chemopreventive Properties

Background: Phenethyl isothiocyanate (PEITC), the most comprehensively studied aromatic isothiocyanate,has been shown to act as an anti-cancer agent mainly through modulation of biotransformation enzymesresponsible for metabolizing carcinogens in the human body. Humans are often exposed to carcinogenic factors,some of which through the diet, such as polycyclic aromatic hydrocarbon benzo[a]pyrene via the consumptionof over-cooked meats. Inhibition of the enzymes responsible for the bioactivation of this carcinogen, for exampleCYP1A1, the major enzyme required for polycyclic aromatic hydrocarbons (PAHs) bioactivation, is recognizedas a chemoprevention strategy. Objective: To evaluate the inhibitory effects of PEITC against benzo[a]pyreneinducedrise in rat liver CYP1A1 mRNA and apoprotein levels. Materials and Methods: Precision cut rat liverslices were treated with benzo[a]pyrene at 1 and 5 μM in the presence of PEITC (1-25 μM) for 24 hours, followedby determination of CYP1A1 mRNA and apoprotein levels using quantitative polymerase chain reaction andimmunoblotting. Results: Findings revealed that PEITC inhibited benzo[a]pyrene-induced rise in rat liverCYP1A1 mRNA in a dose-dependent manner as well as the apoprotein levels of CYP1A. Conclusions: It wasdemonstrated that PEITC can directly inhibit the bioactivation of benzo[a]pyrene, indicating chemopreventivepotential.     

CYP1A1 genotype-selective inhibition of benzo[a]pyrene activation by quercetin

Epidemiological studies suggest that food rich in quercetin and naringin may protect against certain types of lung cancer, and that genotype dependent inhibition of cytochrome P450 1A1 (CYP1A1)-mediated bioactivation of procarcinogens could be the underlying mechanism. We studied the inhibitory effects of quercetin and naringin on the terminal bioactivation step of benzo[a]pyrene (B[a]P), a member of the major class of lung carcinogens. This reaction (epoxidation of (+/-)-trans-7,8-dihydro-7,8-dihydroxy-B[a]P to the ultimate carcinogenic product, (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2)) was examined using three of the most common allelic variants of human CYP1A1, namely wild-type CYP1A1.1, CYP1A1.2, and CYP1A1.4. Quercetin potently inhibited diolepoxide 2 formation by all CYP1A1 types with IC(50) values between 1.6 and 7.0 microM. The differences between the wild-type enzyme and the variants were statistically highly significant (P < 0.01). Enzyme kinetics revealed quercetin as a mixed-type inhibitor of CYP1A1.1, CYP1A1.2, and CYP1A1.4 with K(i) values of 2.0, 6.4, and 9.3 microM, respectively. Naringin inhibited diolepoxide 2 formation only slightly. Our data support the hypothesis that quercetin may have a stronger chemopreventive effect in individuals carrying wild-type compared with variant CYP1A1 genes. Future studies should consider the influence of P450 polymorphisms on both procarcinogen activation and its inhibition to facilitate the development of genotype-specific chemoprevention regimes.     

Tumorigenic activity of non-alternant polynuclear aromatic hydrocarbons in newborn mice

The tumorigenic activity of benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, and indeno-[1,2,3-cd]pyrene was evaluated in newborn CD-1 mice. The total doses of these non-alternant polycyclic aromatic hydrocarbons employed in this study ranged from 0.5 to 2.1 mumol per mouse. The results of this assay indicate that both benzo[b]fluoranthene and benzo[j]fluoranthene exhibit significant tumorigenic activity. In contrast to these results, neither benzo[k]fluoranthene nor indeno[1,2,3-cd]pyrene were tumorigenic under these assay conditions.     

Metabolic proficiency and benzo[a]pyrene DNA adduct formation in APCMin mouse adenomas and uninvolved mucosa.

Tumour formation may involve interactions between genetic factors and environmental carcinogens. Adenoma formation in APCMin/+ mice is associated homozygous adenomatous polyposis coli (APC) gene mutation, but the effects on carcinogen susceptibility are unknown. This study tests the hypothesis that APCMin/+ adenoma formation is accompanied by changes in metabolic proficiency and carcinogen susceptibility. Cytochrome P450 (CYP)1A1/1A2, glutathione S-transferase (GST)alpha, mu and pi classes and DNA adduct formation were assayed in adenomas and uninvolved mucosa from APCMin/+ mice, before and after benzo[a]pyrene (B[a]P) treatment. In untreated adenomas and mucosa, CYP1A1/1A2 and B[a]P-DNA adducts were undetected but GSTalpha, mu and pi class enzymes were constitutively expressed. In adenomas, B[a]P only induced CYP1A1/1A2 to low level while GSTalpha and pi class enzymes were unaffected. A GST mu band which was absent from mucosa, was induced in adenomas. In mucosa, B[a]P induced CYP1A1/1A2 and GSTalpha and pi, to high levels. B[a]P-DNA adduct levels were 56 +/- 15/10(8) nucleotides (median +/- SE) in adenomas versus 89 +/- 19/10(8) nucleotides in mucosa (P < 0.0001). APCMin adenomas show reduced bioactivation capacity and sustain less DNA damage from B[a]P exposure, than APCMin uninvolved mucosa. These properties could influence mutagenesis and subsequent neoplastic transformation of adenomas.     

Binding of polynuclear aromatic hydrocarbons to the rat 4S cytosolic binding protein: structure-activity relationships

The relative competitive binding affinities of benzo[a]pyrene (B[a]P), benzo[e]pyrene, benzo[g, h, i]perylene, picene, 7,12-dimethylbenz [a]anthracene, 1,2,3,4-dibenz[a]anthracene, 1,2,5,6-dibenz[a]anthracene, perylene, 4H-cyclopenta[d,e,f]-phenanthrene, benz[a] anthracene, triphenylethylene and triptycene for the rat hepatic cytosolic 4S binding protein were determined using [3H]benzo[a]pyrene as the radioligand. With the exception of triphenlethylene, triptycene and 4H-cyclopenta[d,e,f]phenanthrene, the EC50 values for the remainder of these compounds were between 1.25 X 10(-7) and 2.5 X 10(-8) M with 1,2,5,6-dibenz[a]anthracene being the most active ligand. A comparison of the relative cytosolic Ah (9S) receptor binding affinities and aryl hydrocarbon hydroxylase (AHH) induction potencies of these hydrocarbons with their 4S protein binding affinities demonstrated the following: five compounds, namely 1,2,5,6-dibenz[a]-anthracene, 1,2,3,4-dibenz[a]anthracene, picene, benzo[a]pyrene and 3-methylcholanthrene exhibited high to moderate binding affinities for the 4S and 9S cytosolic proteins (EC50 values less than 10(-6) M) and induced AHH in rat hepatoma cells; three compounds, namely perylene, benzo[e]pyrene and benzo[g,h,i]perylene exhibited high affinities for the 4S binding protein (1.25 X 10(-7), 4.4 X 10(-8) and 2.9 X 10(-8) M, respectively) and low affinities (EC50 values greater than 10(-5) M) for the Ah receptor protein; moreover these three compounds did not induce AHH in rat hepatoma H-4-II E cells in culture. These data suggest that the 4S binding protein may not play a significant role in AHH induction although the results do not rule out a function for this protein in the transregulation of AHH and its associated cytochromes P-450.