RESUMEN
Although the 3Rs are broadly applied in nonclinical testing, a better appreciation of the 3Rs is needed in the field of differentiated or value-added pharmaceutical generics because the minor changes in formulation, dosage form, indication, and application route often do not require additional safety testing. The US FDA and the EU EMA have comprehensive regulations for such drugs based on quality, therapeutic equivalence, and safety guidelines. However, no scientific publications on how the concept of replacement and reduction from 3Rs principles can be applied in the safety assessment of differentiated generics were found in the public domain. In this review, we discuss the application of 3Rs in nonclinical testing requirements for differentiated generics. Practical examples are provided in the form of case studies from regulated markets. We highlight the need for utilization of existing data to establish equivalence (differentiated generic vs innovator) in efficacy and safety. The case studies indicate that data requirements from animal experiments have been reduced to a large extent in some major markets without compromising quality and safety. In this context, we also highlight the problem that on a global scale, a true reduction of animal experiments will only be achieved when all countries adopt similar practices.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Medicamentos Genéricos/farmacocinética , Alternativas a las Pruebas en Animales/normas , Formas de Dosificación , Vías de Administración de Medicamentos , Quimioterapia Combinada , Medicamentos Genéricos/administración & dosificación , Medicamentos Genéricos/efectos adversos , Europa (Continente) , Humanos , Equivalencia Terapéutica , Estados Unidos , United States Food and Drug AdministrationRESUMEN
New approach methodologies (NAMs), including in vitro toxicology methods such as human cells from simple cell cultures to 3D and organ-on-a-chip models of human lung, intestine, liver, and other organs, are challenging the traditional "norm" of current regulatory risk assessments. Uncertainty Factors continue to be used by regulatory agencies to account for perceived deficits in toxicology data. With the expanded use of human cell NAMs, the question "Are uncertainty factors needed when human cells are used?" becomes a key topic in the development of 21st-century regulatory risk assessment. M.D., PhD, the coauthor of an article detailing uncertainty factors within the U.S. EPA, and L.E., PhD., Executive Vice President, Science, Emulate, who is involved in developing organ-on-a-chip models, debated the topic. One important outcome of the debate was that in the case of in vitro human cells on a chip, the interspecies (animal to human) uncertainty factor of 10 could be eliminated. However, in the case of the intraspecies (average human to sensitive human), the uncertainty factor of 10, additional toxicokinetic and/or toxicodynamic data or related information will be needed to reduce much less eliminate this factor. In the case of other currently used uncertainty factors, such as lowest observable adverse effect level to no-observed adverse effect level extrapolation, missing important toxicity studies, and acute/subchronic to chronic exposure extrapolation, additional data might be needed even when using in vitro human cells. Collaboration between traditional risk assessors with decades of experience with in vivo data and risk assessors working with modern technologies like organ chips is needed to find a way forward.
Asunto(s)
Incertidumbre , Predicción , Humanos , Técnicas In Vitro , Nivel sin Efectos Adversos Observados , Medición de Riesgo/métodosRESUMEN
Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.
Asunto(s)
Daño del ADN/efectos de los fármacos , Mutágenos/toxicidad , Compuestos Orgánicos/toxicidad , Material Particulado/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Aductos de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Genes p53/efectos de los fármacos , Hígado/efectos de los fármacos , RatasRESUMEN
Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.
Asunto(s)
Pruebas de Mutagenicidad/métodos , Nanoestructuras/toxicidad , Animales , Aberraciones Cromosómicas , Ensayo Cometa , Humanos , Técnicas In Vitro/métodos , Pruebas de Mutagenicidad/normas , MutaciónRESUMEN
Exposure of people to hazardous compounds is primarily through complex environmental mixtures, those that occur through media such as air, soil, water, food, cigarette smoke, and combustion emissions. Microarray technology offers the ability to query the entire genome after exposure to such an array of compounds, permitting a characterization of the biological effects of such exposures. This review summarizes the published literature on the transcriptional profiles resulting from exposure of cells or organisms to complex environmental mixtures such as cigarette smoke, diesel emissions, urban air, motorcycle exhaust, carbon black, jet fuel, and metal ore and fumes. The majority of the mixtures generally up-regulate gene expression, with heme oxygenase 1 and CYP1A1 being up-regulated by all of the mixtures. Most of the mixtures altered the expression of genes involved in oxidative stress response (OH-1, metallothioneins), immune/inflammation response (IL-1b, protein kinase), xenobiotic metabolism (CYP1A1, CYP1B1), coagulation and fibrinolysis (plasminogen activator/inhibitor), proto-oncogenes (FUS1, JUN), heat-shock response (HSP60, HSP70), DNA repair (PCNA, GADD45), structural unit of condensed DNA (Crf15Orf16, DUSP 15), and extracellular matrix degradation (MMP1, 8, 9, 11, 12). Genes involved in aldehyde metabolism, such as ALDH3, appeared to be uniquely modulated by cigarette smoke. Cigarette smoke-exposed populations have been successfully distinguished from control nonexposed populations based on the expression pattern of a subset of genes, thereby demonstrating the utility of this approach in identifying biomarkers of exposure and susceptibility. The analysis of gene-expression data at the pathway and functional level, along with a systems biology approach, will provide a more comprehensive insight into the biological effects of complex mixtures and will improve risk assessment of the same. We suggest critical components of study design and reporting that will achieve this goal.
Asunto(s)
Monitoreo del Ambiente/métodos , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Contaminantes Atmosféricos/análisis , Exposición a Riesgos Ambientales/análisis , Humanos , Emisiones de Vehículos/análisisRESUMEN
Human exposures to polycyclic aromatic hydrocarbon (PAH) occur in complex mixtures. Here, gene expression patterns were investigated using standard reference material (SRM) 1649a (urban dust). MCF-7 cells were exposed to SRM 1649a alone or SRM 1649a with either benzo[a]pyrene (BP) or dibenzo[a,l]pyrene (DBP) for 24 hours. Global analyses of the gene expression data revealed alterations of 41 RNA transcripts with at least 2-fold change (signal log ratio = -1 or >/= 1) in response to SRM 1649a exposure. Increase in expression of cytochrome P450 (CYP) genes was observed in response to BP exposure (CYP1A1 and CYP1B1; signal log ratio of 4.7 and 2.5, respectively). An additive induction of CYP1A1 and CYP1B1 was observed with cotreatment of SRM 1649a and BP. On the contrary, no change in gene expression of CYP1A1 and CYP1B1 was observed when the cells were exposed to DBP. Furthermore, to study the effect of complex PAH mixtures on the metabolic activation of carcinogenic PAH to DNA-binding derivatives and to relate this with gene expression studies, PAH-DNA adduct formation was determined. SRM 1649a decreased the total level of BP-DNA adducts in comparison with BP alone. No significant difference in adduct levels was observed in response to either DBP alone or in combination with SRM 1649a. These results provide a transcriptional signature for chemical carcinogen exposure; in addition, they suggest a major factor in carcinogenic activity of PAH within complex mixtures is their ability to promote or inhibit the activation of carcinogenic PAH by the induction of CYP enzymes.
Asunto(s)
Benzo(a)pireno/farmacología , Benzopirenos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Polvo , Expresión Génica/efectos de los fármacos , Apoptosis/genética , Benzo(a)pireno/química , Benzo(a)pireno/farmacocinética , Benzopirenos/química , Benzopirenos/farmacocinética , Biotransformación , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/enzimología , Carcinógenos/farmacocinética , Carcinógenos/farmacología , Ciclo Celular/genética , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Aductos de ADN/biosíntesis , Daño del ADN , Reparación del ADN/genética , Humanos , IsoenzimasRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.
Asunto(s)
Crisenos/toxicidad , Daño del ADN/efectos de los fármacos , Neoplasias/genética , Neoplasias/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Crisenos/química , Crisenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Aductos de ADN/metabolismo , Humanos , Estructura MolecularRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens present in the atmosphere from combustion sources such as cigarette smoke, diesel exhaust, residential heating processes, and industrial coke production. To date, dibenzo[a,l]pyrene (DBP) has been found to be the strongest tumor-initiating PAH ever tested in rodent skin and mammary tumor models. Here we show for the first time that systemic exposure to DBP causes DNA damage in mouse lung tissue. C57BL/6 mice were gavaged with 1, 5 or 20 mg DBP/kg body weight, daily for 10 days. Toxicity of DBP was revealed by a decrease in body and organ weight of mice while no apparent cell death was observed on P815 mastocytoma cells (allograft model) in vitro. However, treatment of P815 cells in vitro with the ultimate carcinogenic metabolite of DBP, the fjord region (-)-anti-11,12-diol 13,14-epoxide [(-)-anti-DBPDE], resulted in the total loss of cell viability. Lungs from the animals were removed and subjected to DBP-DNA adduct analysis. A dose dependent adduct formation was revealed by 33P-postlabeling analysis of DNA from lung tissue. The majority of DNA adducts formed in lungs of mice after systemic exposure to DBP were contributed by (-)-anti-DBPDE. The data from this in vivo model are consistent with previous metabolic activation results obtained with DBP in human cells in culture.
Asunto(s)
Benzopirenos/toxicidad , Aductos de ADN/análisis , Pulmón/efectos de los fármacos , Animales , Benzopirenos/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Femenino , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
Humans are exposed to complex mixtures of polycyclic aromatic hydrocarbons in the atmosphere. We examined the long term effects of a standard reference material (SRM) 1649a over time on the metabolic activation and DNA adduct formation by two carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma derived cell line MCF-7. PAH-DNA adduct analysis, cytochrome P450 (CYP) enzyme activity, CYP1A1 and CYP1B1 protein expression were determined in cells treated with SRM 1649a alone or SRM 1649a with either BP or DBP for 24 to 120 h. Averaging over time, significantly higher levels of DNA adducts were observed in cells treated with BP or DBP alone than in co-treatments with SRM 1649a and BP or DBP. Ethoxyresorufin O-deethylase assay indicated a significant increase in activity in cells treated with BP alone and co-treated with SRM1649a in comparison to other treatment groups. Induction of CYP1A1 and CYP1B1 protein expression was observed by immunoblots in cells treated with BP alone or in co-treatments of SRM 1649a and BP or DBP. These data demonstrate the influence of the components of SRM 1649a in inhibiting the activation of BP or DBP by CYP enzymes in the formation of DNA adducts. It also suggests that the carcinogenic activity of PAH within a complex mixture may vary with composition and activation of the components present in the complex mixture.
Asunto(s)
Benzo(a)pireno/farmacocinética , Benzopirenos/farmacocinética , Neoplasias de la Mama/metabolismo , Carcinógenos/farmacocinética , Mezclas Complejas/farmacocinética , Polvo , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Benzo(a)pireno/toxicidad , Benzopirenos/toxicidad , Biotransformación , Neoplasias de la Mama/tratamiento farmacológico , Carcinógenos/toxicidad , Línea Celular Tumoral , Mezclas Complejas/toxicidad , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1B1 , ADN/efectos de los fármacos , ADN/metabolismo , Aductos de ADN/biosíntesis , Aductos de ADN/efectos de los fármacos , Combinación de Medicamentos , Humanos , Microsomas/efectos de los fármacos , Microsomas/enzimología , Salud UrbanaRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are a class of widespread environmental carcinogens. Most of our knowledge of their mechanisms of metabolic activation to DNA-binding "ultimate carcinogenic" metabolites has come from analysis of the DNA interaction products formed by these highly reactive intermediates. Studies of their role in forming DNA-binding intermediates identical to those formed in vivo from the PAH itself have also allowed identification of the particular cytochrome P450 enzymes involved in activating various structural classes of carcinogenic PAHs. It has been established that PAHs, after metabolic activation in vivo, are capable of inducing mutations in oncogenes and, by inducing multiple mutations, may result in tumors. PAHs also cause changes in cellular gap-junction communication similar to those caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Thus, PAHs may also act through a promotional mechanism in addition to serving as tumor initiators. Previous studies on these mechanisms are described and summarized.
Asunto(s)
Carcinógenos Ambientales/metabolismo , Aductos de ADN/metabolismo , ADN/metabolismo , Modelos Biológicos , Hidrocarburos Policíclicos Aromáticos/metabolismo , Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Carcinógenos Ambientales/química , Conexinas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/genética , Mutación/genética , Hidrocarburos Policíclicos Aromáticos/químicaRESUMEN
Diesel exhaust particles (DEP) are a major source of air-borne pollution and are linked to increased risk of disease including lung cancer. Here we investigated effects of exposure to DEP on the frequency of DNA deletions, levels of oxidative DNA damage and DNA adduct formation during embryonic development in mice. Pregnant dams were orally exposed to various doses of DEP (500, 250, 125, 62.5, 31.25 mg/kg/day) at embryonic days 10.5-15.5. We determined the frequency of 70 kb DNA deletions spanning exons 6-18 at the p(un) allele that results in black-pigmented spots in the unpigmented retinal pigment epithelium in the eyes of p(un)/p(un) offspring mice. DEP caused a significant increase in the frequency of DNA deletions. Levels of 8-OH deoxyguanosine indicating oxidative DNA damage were within the limits of the unexposed mouse embryos. 33P post-labeling analysis revealed very low levels of DNA adducts in the embryo tissue. Thus, transplacental exposure to DEP resulted in a significant increase in the frequency of DNA deletions in the mouse fetus and such genetic alterations in the offspring may have pathological consequences later in life.
Asunto(s)
ADN/efectos de los fármacos , Placenta/metabolismo , Emisiones de Vehículos , Animales , ADN/genética , Desarrollo Embrionario/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos C57BL , EmbarazoRESUMEN
The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets.
Asunto(s)
Inmunidad Innata , Inmunidad Mucosa , Modelos Inmunológicos , Sus scrofa/inmunología , Animales , Animales Recién Nacidos , Biomarcadores/sangre , Biomarcadores/metabolismo , Cruzamientos Genéticos , Citocinas/sangre , Femenino , Citometría de Flujo/veterinaria , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/crecimiento & desarrollo , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Inmunohistoquímica/veterinaria , Inmunofenotipificación/veterinaria , Masculino , Michigan , Membrana Mucosa/citología , Membrana Mucosa/crecimiento & desarrollo , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Estabilidad Proteica , Reproducibilidad de los Resultados , Sus scrofa/sangre , Sus scrofa/crecimiento & desarrollo , Sus scrofa/metabolismo , Pruebas de ToxicidadRESUMEN
Human exposure to polycyclic aromatic hydrocarbons (PAHs) occurs through complex mixtures. The National Institute of Standards and Technology has established standard reference materials (SRMs) for selected PAH mixtures that are composed of carcinogenic, noncarcinogenic, and weakly carcinogenic compounds, such as those derived from coal tar (SRM 1597), atmospheric particulate matter (SRM 1649), and diesel particulate matter (SRM 1650). To study the effects of PAHs with different carcinogenic potential in complex mixtures, and to investigate the metabolic activation of noncarcinogenic and weakly carcinogenic PAHs to DNA-binding derivatives, artificial mixtures (1597H, 1649H, and 1650H) were prepared in the laboratory. These artificial mixtures contained the same relative ratios of noncarcinogenic and weakly carcinogenic PAHs present in SRM 1597, SRM 1649, and SRM 1650. The human mammary carcinoma-derived cell line MCF-7 was treated with these artificial mixtures and analyzed for PAH-DNA adduct formation and the induction of cytochrome P450 (CYP) enzymes. We found that the artificial mixtures formed lower but detectable levels of DNA adducts 24 and 48 hr after treatment than benzo[a]pyrene. Induction of CYP enzyme activity was measured by the ethoxyresorufin-O-deethylase assay, and the expression of CYP1A1 and CYP1B1 was confirmed by immunoblots. Both noncarcinogenic and weakly carcinogenic PAHs present in the artificial mixtures have the ability to induce CYP1A1 and CYP1B1 in MCF-7 cells and contribute to DNA binding. Therefore, it is necessary to take into account the noncarcinogenic and weakly carcinogenic PAHs present in environmental mixtures in assessing the potential risk associated with human exposure.
Asunto(s)
Alquitrán/química , Polvo/análisis , Compuestos Policíclicos/toxicidad , Emisiones de Vehículos/análisis , Línea Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Humanos , Microsomas/efectos de los fármacos , Microsomas/enzimología , Compuestos Policíclicos/análisisRESUMEN
The polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) is an exceptionally potent carcinogen. Its direct DNA-reactive metabolite, the fjord region (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide [(-)-anti-DB[a,l]PDE], was used to investigate induction of mutations in the coding region of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster V79 cells. Cells exposed to 1-10 nM (-)-anti-DB[a,l]PDE exhibited a close dose-responsive increase in the frequency of mutant clones resistant to 6-thioguanine. RNA was isolated from mutant clones and cDNAs were prepared by reverse transcription. The coding region of the cDNA of the Hprt gene was amplified by polymerase chain reaction and sequenced. Analysis of the DNA base sequence changes induced by (-)-anti-DB[a,l]PDE indicated that base substitutions were the most prevalent mutations, followed by exon deletions. Among the groups of V79 cells treated with low concentrations of (-)-anti-DB[a,l]PDE, most displayed high selectivity for both A:T-->T:A transversions and A:T-->G:C transitions, while cells exposed to a higher dose (10 nM) formed predominantly G:C-->T:A transversions. Also, the number of base substitutions per mutant clone increased with dose. In general, the mutation profiles induced by (-)-anti-DB[a,l]PDE exhibited a wide spectrum; in addition to base substitutions, deletions, insertions, frameshift mutations, as well as tandem mutations were detected. Analysis of the DNA adduct levels induced by (-)-anti-DB[a,l]PDE revealed that a concentration-dependent increase in the level of adduct formation preceded the concentration-dependent increase in mutational events in these cells and that an increasing proportion of DNA adducts at deoxyadenosine were formed with dose. The results of this study demonstrate a correspondence between the concentration and types of DNA adducts and the frequency and types of mutations induced by (-)-anti-DB[a,l]PDE in V79 cells.
Asunto(s)
Benzopirenos/farmacología , Aductos de ADN/metabolismo , Compuestos Epoxi/farmacología , Hipoxantina Fosforribosiltransferasa/genética , Mutación/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Cricetinae , Cartilla de ADN/química , ADN Complementario/genética , ADN Complementario/metabolismo , Compuestos Epoxi/antagonistas & inhibidores , Exones/genética , Eliminación de Gen , Hipoxantina Fosforribosiltransferasa/metabolismo , Reacción en Cadena de la Polimerasa , Tioguanina/farmacologíaRESUMEN
DNA adducts generated by carcinogenic chemicals reflects human exposure and DNA adducts are related to tumor formation. Most chemical carcinogens require activation to reactive intermediates that bind to nucleophilic centers in proteins and nucleic acids thereby forming covalent adducts. Also, many of the chemicals considered carcinogenic for humans form covalent DNA adducts. Therefore, such DNA damage is generally considered to be causative and linked to tumor formation. In this article we have summarized the work done for many years on the role of DNA adduct formation as an indicator of their carcinogenicity. We have also addressed the important role for measurement of DNA adducts in studies with potential chemopreventive agents for which it is central to have a marker that can be measured more rapidly than changes in cancer incidence.
Asunto(s)
Carcinógenos/metabolismo , Quimioprevención/métodos , Aductos de ADN/análisis , Mutagénesis , Animales , Biomarcadores de Tumor/análisis , Daño del ADN , ADN de Neoplasias/genética , Humanos , Neoplasias/genéticaRESUMEN
Octenyl succinic anhydride (OSA)-modified starch functions as both an emulsifier and emulsion stabilizer in foods, and is intended for use in infant formula, follow-on formula, and formulae for special medical purposes. These formulae predominantly include extensively hydrolyzed protein or free amino acids, rather than intact protein, which otherwise would provide emulsifying functionality. The study objectives were to evaluate (1) the safety of OSA-modified starch after three weeks of administration to neonatal farm piglets, beginning 2 days after birth and (2) the impact of OSA-modified starch on piglet growth. OSA-modified starch was added to formula at concentrations of 2, 4, and 20 g/L. The vehicle control, low-dose, and mid-dose diets were supplemented with Amioca™ Powder to balance the nutritional profiles of all formulations. There were no test article-related effects of any diet containing OSA-modified starch on piglet growth and development (clinical observations, body weight, feed consumption), or clinical pathology parameters (hematology, clinical chemistry, coagulation, urinalysis). In addition, there were no adverse effects at terminal necropsy (macro- and microscopic pathology evaluations). Therefore, dietary exposure to OSA-modified starch at concentrations up to 20 g/L was well tolerated by neonatal farm piglets and did not result in adverse health effects or impact piglet growth.
Asunto(s)
Seguridad de Productos para el Consumidor , Inocuidad de los Alimentos , Almidón/análogos & derivados , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Dieta , Relación Dosis-Respuesta a Droga , Heces/química , Femenino , Masculino , Almidón/administración & dosificación , Almidón/toxicidad , PorcinosRESUMEN
This unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real-time PCR (qRT-PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7-ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3',5'-Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC50 of 3 nM for EROD and ~90 nM for E2 4-hydroxylation. Binding studies with purified CYP1B1 suggests that TMS interferes in the proximity of the heme region of CYP1B1 with high affinity. Compared to other potent inhibitors such as α-naphthoflavone, which is a known CYP1 family inhibitor with no selectivity between CYP1B1 and CYP1A2, TMS is ~50- and 520-fold selective for inhibition of CYP1B1 when compared to CYP1A1 and CYP1A2, respectively. Thus, TMS can serve as a helpful chemical scalpel for dissecting CYP1B1 activity from the overall activity of CYP1 family members against ethoxyresorufin.