RESUMO
The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.
Assuntos
Desenvolvimento de Medicamentos , Terapia Genética , Distribuição Tecidual , Reação em Cadeia da PolimeraseRESUMO
Humans are variously and continuously exposed to a wide range of different DNA-damaging agents, some of which are classed as carcinogens. DNA damage can arise from exposure to exogenous agents, but damage from endogenous processes is probably far more prevalent. That said, epidemiological studies of migrant populations from regions of low cancer risk to high cancer risk countries point to a role for environmental and/or lifestyle factors playing a pivotal part in cancer aetiology. One might reasonably surmise from this that carcinogens found in our environment or diet are culpable. Exposure to carcinogens is associated with various forms of DNA damage such as single-stand breaks, double-strand breaks, covalently bound chemical DNA adducts, oxidative-induced lesions and DNA-DNA or DNA-protein cross-links. This review predominantly concentrates on DNA damage induced by the following carcinogens: polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, mycotoxins, ultraviolet light, ionising radiation, aristolochic acid, nitrosamines and particulate matter. Additionally, we allude to some of the cancer types where there is molecular epidemiological evidence that these agents are aetiological risk factors. The complex role that carcinogens play in the pathophysiology of cancer development remains obscure, but DNA damage remains pivotal to this process.
Assuntos
Carcinógenos/química , Dano ao DNA , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Aminas/efeitos adversos , Animais , Ácidos Aristolóquicos/efeitos adversos , Benzo(a)pireno/efeitos adversos , Reagentes de Ligações Cruzadas/química , DNA , Reparo do DNA , Dieta , Feminino , Humanos , Inflamação , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Masculino , Neoplasias/induzido quimicamente , Neoplasias/genética , Estresse Oxidativo , Neoplasias da Próstata/induzido quimicamente , Neoplasias da Próstata/genética , Fumar/efeitos adversos , Raios Ultravioleta/efeitos adversos , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/genéticaRESUMO
The nucleoside reverse transcriptase inhibitor zidovudine (AZT) induces genotoxic damage that includes centrosomal amplification (CA > 2 centrosomes/cell) and micronucleus (MN) formation. Here we explored these end points in mice deficient in DNA repair and tumor suppressor function to evaluate their effect on AZT-induced DNA damage. We used mesenchymal-derived fibroblasts cultured from C57BL/6J mice that were null and wild type (WT) for Xpa, and WT, haploinsufficient and null for p53 (6 different genotypes). Dose-responses for CA formation, in cells exposed to 0, 10, and 100 µM AZT for 24 hr, were observed in all genotypes except the Xpa((+/+)) p53((+/-)) cells, which had very low levels of CA, and the Xpa((-/-)) p53((-/-)) cells, which had very high levels of CA. For CA there was a significant three-way interaction between Xpa, p53, and AZT concentration, and Xpa((-/-)) cells had significantly higher levels of CA than Xpa((+/+)) cells, only for p53((+/-)) cells. In contrast, the MN and MN + chromosomes (MN + C) data showed a lack of AZT dose response. The Xpa((-/-)) cells, with p53((+/+)) or ((+/-)) genotypes, had levels of MN and MN + C higher than the corresponding Xpa((+/+)) cells. The data show that CA is a major event induced by exposure to AZT in these cells, and that there is a complicated relationship between AZT and CA formation with respect to gene dosage of Xpa and p53. The loss of both genes resulted in high levels of damage, and p53 haploinsufficicency strongly protected Xpa((+/+)) cells from AZT-induced CA damage.
Assuntos
Centrossomo/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Zidovudina/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/genética , Relação Dose-Resposta a Droga , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes para Micronúcleos , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.
Assuntos
Adutos de DNA/metabolismo , DNA/metabolismo , Tamoxifeno/metabolismo , Útero/metabolismo , Animais , DNA/química , Adutos de DNA/efeitos adversos , Adutos de DNA/química , Endométrio/metabolismo , Erythrocebus patas , Feminino , Humanos , Miométrio/metabolismo , Tamoxifeno/químicaRESUMO
Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.
Assuntos
Fator de Crescimento Epidérmico/genética , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento Derivado de Plaquetas/genética , Receptores de Hidrocarboneto Arílico/fisiologia , Compostos Azo/farmacologia , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Neoplasias de Cabeça e Pescoço , Humanos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Pirazóis/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. In addition, our recent studies implicate the AHR in the regulation of the expression of several growth factors in established tumor cell lines. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA (RA-FLS). Treatment with the AHR antagonist GNF351 inhibits cytokine-induced expression of vascular endothelial growth factor-A (VEGF-A), epiregulin, amphiregulin, and basic fibroblast growth factor mRNA through an AHR-dependent mechanism in both RA-FLS and FLS. Secretion of VEGF-A and epiregulin from RA-FLS was also inhibited upon GNF351 treatment. RA-FLS cell migration, along with cytokine-induced RA-FLS cell proliferation, was significantly attenuated by GNF351 exposure. Treatment of RA-FLS with GNF351 mitigated cytokine-mediated expression of matrix metalloproteinase-2 and -9 mRNA and diminished the RA-FLS invasive phenotype. These findings indicate that inhibition of AHR activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating growth factor release; FLS proliferation, migration, and invasion; and inflammatory activity.
Assuntos
Antirreumáticos/farmacologia , Artrite Reumatoide/tratamento farmacológico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Membrana Sinovial/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Anfirregulina , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Família de Proteínas EGF , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Gelatinases/antagonistas & inibidores , Gelatinases/genética , Gelatinases/metabolismo , Inativação Gênica , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Terapia de Alvo Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Purinas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Elementos de Resposta/efeitos dos fármacos , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVES: Rheumatoid Arthritis (RA) is a chronic inflammatory disease of unclear aetiology, which is associated with inflamed human fibroblast-like synoviocytes (HFLS). Epidemiological studies have identified a positive correlation between tobacco smoking (a rich source of aryl hydrocarbon receptor (AHR) agonists) and aggressive RA phenotype. Thus, we hypothesise that antagonism of AHR activity by a potent AHR antagonist GNF351 can attenuate the inflammatory phenotype of HFLS-RA cells. METHODS: Quantitative PCR was used to examine IL1B-induced mRNA expression in primary HFLS-RA cells. A structurally diverse AHR antagonist CH223191 and transient AHR repression using AHR small interfering RNA (siRNA) in primary HFLS-RA cells were used to demonstrate that effects observed by GNF351 are AHR-mediated. The levels of PTGS2 were determined by western blot and secretory cytokines such as IL1B and IL6 by ELISA. Chromatin-immunoprecipitation was used to assess occupancy of the AHR on the promoters of IL1B and IL6. RESULTS: Many of the chemokine and cytokine genes induced by IL1B in HFLS-RA cells are repressed by co-treatment with GNF351 at both the mRNA and protein level. Pretreatment of HLFS-RA cells with CH223191 or transient gene ablation of AHR by siRNA confirmed that the effects of GNF351 are AHR-mediated. GNF351 inhibited the recruitment of AHR to the promoters of IL1B and IL6 confirming occupancy of AHR at these promoters is required for enhanced inflammatory signalling. CONCLUSIONS: These data suggest that AHR antagonism may represent a viable adjuvant therapeutic strategy for the amelioration of inflammation associated with RA.
Assuntos
Artrite Reumatoide/patologia , Fibroblastos/efeitos dos fármacos , Indóis/farmacologia , Mediadores da Inflamação/metabolismo , Purinas/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Artrite Reumatoide/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
The aryl hydrocarbon receptor (AhR) has been shown to play a role in an increasing number of cellular processes. Recent reports have linked the AhR to cell proliferation, cytoskeletal arrangement, and tumor invasiveness in various tumor cell types. The AhR plays a role in the de-repression of the interleukin (IL)6 promoter in certain tumor cell lines, allowing for increased transcriptional activation by cytokines. Here, we show that there is a significant level of constitutive activation of the AhR in cells isolated from patients with head and neck squamous cell carcinoma (HNSCC). Constitutive activation of the AhR in HNSCCs was blocked by antagonist treatment, leading to a reduction in IL6 expression. In addition, the AhR exhibits a high level of expression in HNSCCs than in normal keratinocytes. These findings led to the hypothesis that the basal AhR activity in HNSCCs plays a role in the aggressive phenotype of these tumors and that antagonist treatment could mitigate this phenotype. This study provides evidence that antagonism of the AhR in HNSCC tumor cells, in the absence of exogenous receptor ligands, has a significant effect on tumor cell phenotype. Treatment of these cell lines with the AhR antagonists 6, 2', 4'-trimethoxyflavone, or the more potent GNF351, decreased migration and invasion of HNSCC cells and prevented benzo[a]pyrene-mediated induction of the chemotherapy efflux protein ABCG2. Thus, an AhR antagonist treatment has been shown to have therapeutic potential in HNSCCs through a reduction in aggressive cell phenotype.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Humanos , Indóis , Interleucina-6/genética , Interleucina-6/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Fenótipo , Purinas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.
Assuntos
Benzo(a)pireno/toxicidade , Adutos de DNA/metabolismo , Fígado/efeitos dos fármacos , NADPH-Ferri-Hemoproteína Redutase/deficiência , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Benzo(a)pireno/metabolismo , Benzo(a)pireno/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromos b5/metabolismo , Dano ao DNA , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Imuno-Histoquímica , Fígado/enzimologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genéticaRESUMO
We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(-/-)p53(+/-) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N (2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(-/-)p53(+/-) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(-/-)p53(+/-) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(-/-)p53(+/-) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP-DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(-/-)p53(+/-) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH-DNA adduct levels consistently in human organs.
Assuntos
Benzo(a)pireno/toxicidade , Clorofilídeos/farmacologia , Adutos de DNA/efeitos dos fármacos , Reparo do DNA/genética , Haploinsuficiência , Proteína Supressora de Tumor p53/fisiologia , Proteína de Xeroderma Pigmentoso Grupo A/fisiologia , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Animais , Antimutagênicos/farmacologia , Carcinógenos/metabolismo , Cromatografia Líquida de Alta Pressão , Adutos de DNA/metabolismo , Dano ao DNA/genética , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espectrometria de Massas em TandemRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are combustion products of organic materials, mixtures of which contain multiple known and probable human carcinogens. PAHs occur in indoor and outdoor air, as well as in char-broiled meats and fish. Human exposure to PAHs occurs by inhalation, ingestion and topical absorption, and subsequently formed metabolites are either rendered hydrophilic and excreted, or bioactivated and bound to cellular macromolecules. The formation of PAH-DNA adducts (DNA binding products), considered a necessary step in PAH-initiated carcinogenesis, has been widely studied in experimental models and has been documented in human tissues. This review describes immunohistochemistry (IHC) studies, which reveal localization of PAH-DNA adducts in human tissues, and semi-quantify PAH-DNA adduct levels using the Automated Cellular Imaging System (ACIS). These studies have shown that PAH-DNA adducts concentrate in: basal and supra-basal epithelium of the esophagus, cervix and vulva; glandular epithelium of the prostate; and cytotrophoblast cells and syncitiotrophoblast knots of the placenta. The IHC photomicrographs reveal the ubiquitous nature of PAH-DNA adduct formation in human tissues as well as PAH-DNA adduct accumulation in specific, vulnerable, cell types. This semi-quantative method for PAH-DNA adduct measurement could potentially see widespread use in molecular epidemiology studies.
Assuntos
Carcinógenos/análise , Adutos de DNA/análise , Imuno-Histoquímica/métodos , Hidrocarbonetos Policíclicos Aromáticos/análise , Carcinógenos/metabolismo , Adutos de DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica/instrumentação , Masculino , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Distribuição TecidualRESUMO
Three classes of DNA damage were assessed in human placentas collected (2000-2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by (32)P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7ß,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49-312 PAH-DNA adducts/10(8) nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7-8.6 stable/bulky DNA adducts/10(8) nucleotides and 0.6-47.2 AB sites/10(5) nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi.
Assuntos
Dano ao DNA , Fumar/efeitos adversos , República Tcheca , Adutos de DNA/toxicidade , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Soros Imunes , Imuno-Histoquímica , Técnicas In Vitro , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , GravidezRESUMO
Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4muM) in the absence or presence of chlorophyllin (5muM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (p<0.005) and CYP1B1 expression in 17/20 NHMEC strains (p<0.005). Maximum percent reductions of CYP1A1 and CYP1B1 gene expression and BPdG adduct formation were observed when cells were pre-dosed with chlorophyllin followed by administration of the carcinogen with chlorophyllin (p<0.005 for CYP1A1 and CYP1B1 expression and p<0.0005 for BPdG adducts). Therefore, chlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Benzo(a)pireno/farmacologia , Clorofilídeos/farmacologia , Citocromo P-450 CYP1A1/genética , Adutos de DNA , Glândulas Mamárias Humanas/efeitos dos fármacos , Citocromo P-450 CYP1B1 , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Carcinogenic polycylic aromatic hydrocarbons can alter immune responses. Changes in immune response gene expression profiles in multiple human mammary cell strains exposed to benzo(alpha)pyrene (BP) (4 microM) in vitro, in the presence or absence of chlorophyllin (5 microM), were observed using Affymetrix gene arrays. Expressions of five immune response genes were altered ~3.0-fold by BP exposure and 24 genes by BP in the presence chlorophyllin. In silico pathway analysis revealed altered immune response genes form interactive gene networks with many cellular processes, suggesting their role in a complex multigenic response to toxins. Additionally, it was suggestive of the possible immunomodulatory potential of chlorophyllin apart from various other well-documented mechanisms of action. Gene expression matrices revealed consistent alteration patterns involving IL1B, SECTM1 and CXCL14 on exposure to BP, and IL1RN, CD86, IF144 and GIP2 in the presence of chlorophyllin and BP, suggesting some of these genes might constitute putative immune response biomarkers of PAH exposure. This study has therefore identified a battery of potential immune response biomarkers of PAH exposure, amidst several genes, for future validation studies.
Assuntos
Benzo(a)pireno/toxicidade , Biomarcadores/metabolismo , Clorofilídeos/farmacologia , Perfilação da Expressão Gênica , Genes MHC da Classe II/genética , Imunidade Celular/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Antimutagênicos/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Glândulas Mamárias Humanas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Studies of migrant populations suggest that dietary and/or environmental factors play a crucial role in the etiology of prostatic adenocarcinoma (CaP). The human prostate consists of the peripheral zone (PZ), transition zone (TZ), and central zone (CZ); CaP occurs most often in the PZ. METHODS: To investigate the notion that an underlying differential expression of phase I/II genes, and/or the presence of polycyclic aromatic hydrocarbon (PAH)-DNA adducts might explain the elevated PZ susceptibility, we examined prostate tissues (matched tissue sets consisting of PZ and TZ) from men undergoing radical retropubic prostatectomy for CaP (n = 26) or cystoprostatectomy (n = 1). Quantitative gene expression analysis was employed for cytochrome P450 (CYP) isoforms CYP1A1, CYP1B1, and CYP1A2, as well as N-acetyltransferase 1 and 2 (NAT1 and NAT2) and catechol-O-methyl transferase (COMT). RESULTS: CYP1B1, NAT1, and COMT were expressed in all tissue sets; levels of CYP1B1 and NAT1 were consistently higher in the PZ compared to TZ. Immunohistochemistry confirmed the presence of CYP1B1 (nuclear-associated and primarily in basal epithelial cells) and NAT1. Normal tissue from 23 of these aforementioned 27 matched tissue sets was analyzed for PAH-DNA adduct levels using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). PAH-DNA adduct levels were highest in glandular epithelial cells, but a comparison of PZ and TZ showed no significant differences. CONCLUSION: Although expression of activating and/or detoxifying enzymes may be higher in the PZ, PAH-DNA adduct levels appear to be similar in both zones. Therefore, factors other than PAH-DNA adducts may be responsible for promotion of tumor formation in the human prostate.
Assuntos
Adenocarcinoma/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Catecol O-Metiltransferase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Adutos de DNA/metabolismo , Isoenzimas/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/patologia , Hidrocarboneto de Aril Hidroxilases , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Citocromo P-450 CYP1B1 , Suscetibilidade a Doenças , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/patologiaRESUMO
Benzo(a)pyrene (BP) exposure causes alterations in gene expression in normal human mammary epithelial cells (NHMECs). This study used Affymetrix Hu-Gene133A arrays, with 14,500 genes represented, to evaluate modulation of BP-induced gene expression by chlorophyllin in six NHMEC strains derived from different donors. A major goal was to seek potential biomarkers of carcinogen exposure and how they behave in the presence of a chemopreventive agent. NHMECs (passage 6 and 70% confluence) were exposed for 24h to either vehicle control, or BP, or chlorophyllin followed by BP and chlorophyllin together. BP exposure resulted in approximately 3-fold altered expression of 49 genes in at least one of the six NHMEC strains. When cells were exposed to chlorophyllin pre-treatment followed by BP plus chlorophyllin, expression of 125 genes was similarly altered. Genes in the functional categories of xenobiotic metabolism, cell signaling, cell motility, cell proliferation, cellular transcription, metabolism, cell cycle control, apoptosis and DNA repair were identified. Only CYP1B1 and ALDH1A3 were consistently up-regulated by approximately 3-fold in most of the cell strains (at least 4) when exposed to BP. Cluster analysis identified a suite of 13 genes induced by BP where induction was mitigated in the presence of chlorophyllin. Additionally, cluster analysis identified a suite of 16 genes down-regulated by BP where induction was partially restored in the presence of chlorophyllin.