RESUMEN
Tributyltin (TBT) and dioxin-like polychlorinated biphenyls (PCBs) are environmental contaminants that are highly toxic to fish and co-occur in New Bedford Harbor (NBH), an estuarine Superfund site located in Massachusetts, USA. Atlantic killifish (Fundulus heteroclitus) that reside in NBH (and other highly contaminated sites along the east coast of the United States) have developed resistance to activation of the aryl hydrocarbon receptor (AHR) pathway and the toxicity of dioxin-like chemicals, such as 3,3',4,4',5-pentachlorobiphenyl, PCB126. In many biological systems, TBT disregulates adipose and bone development via the PPARγ-RXR pathway; AHR activation also disrupts adipose and bone homeostasis, potentially through molecular crosstalk between AHR and PPARγ. However, little is known about how co-exposure and the interaction of these pathways modulate the toxicological effects of these contaminants. Here, we tested the hypotheses that TBT would induce teratogenesis in killifish via activation of PPARγ and that PCB126 co-exposure would suppress PPARγ pathway activation in PCB-sensitive killifish from a reference site (Scorton Creek, SC, PCB-sensitive) but not in PCB-tolerant NBH killifish. Killifish embryos from both populations exposed to TBT (50 and 100â¯nM) displayed caudal fin deformities. TBT did not change the expression of pparg or its target genes related to adipogenesis (fabp11a and fabp1b) in either population. However, expression of osx/sp7, an osteoblast marker gene, and col2a1b, a chondroblast marker gene, was significantly suppressed by TBT only in SC killifish. An RXR-specific agonist, but not a PPARγ-specific agonist, induced caudal fin deformities like those observed in TBT-treated embryos. PCB126 did not induce caudal fin deformities and did not exacerbate TBT-induced fin deformities. Further, PCB126 increased expression of pparg in SC embryos and not NBH embryos, but did not change the expression of fabp1b. Taken together, these results suggest that in killifish embryos the PPARγ pathway is regulated in part by AHR, but is minimally active at least in this early life stage. In killifish, RXR activation, rather than PPARγ activation, appears to be the mechanism by which TBT induces caudal fin teratogenicity, which is not modulated by AHR responsiveness.
Asunto(s)
Aletas de Animales/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Fundulidae , PPAR gamma/metabolismo , Bifenilos Policlorados/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Aletas de Animales/anomalías , Animales , Resistencia a Medicamentos/efectos de los fármacos , Sinergismo Farmacológico , Embrión no Mamífero/anomalías , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Massachusetts , PPAR gamma/genética , Receptor Cross-Talk , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.
Asunto(s)
Cisplatino/farmacología , Aductos de ADN/efectos de los fármacos , Aductos de ADN/genética , ADN Polimerasa Dirigida por ADN , Secuencia de Bases , Sitios de Unión , Cisplatino/metabolismo , Aductos de ADN/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Humanos , Mapeo Físico de Cromosoma , Plásmidos/genética , Plásmidos/metabolismo , Platino (Metal)/metabolismo , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Telómero/genética , Moldes Genéticos , Proteínas Virales/metabolismoRESUMEN
Chemical and biological studies are presented for a new series of platinum(II) antitumor agents that violate the classical structure-activity relationships established for platinum complexes. These new agents, which have demonstrated activity against murine and human tumor systems, are cis-[Pt(NH3)2(Am)Cl]+ cations, in which Am is a derivative of pyridine, pyrimidine, purine, or aniline. Members from this series block simian virus 40 DNA replication in vitro and inhibit the action of DNA polymerases at individual guanine residues in replication mapping experiments. Monoclonal antibodies that bind selectively to cisplatin lesions on calf thymus DNA were used in a competitive enzyme-linked immunosorbent assay study to show that the platinum-triamine complexes do not produce the type of intrastrand cross-links on DNA that are characteristics of cisplatin and analogues with the general formula cis-[Pt(amine)2X2]. These results indicate that cis-[Pt(NH3)2(Am)Cl]+ cations form monofunctional adducts on DNA rather than eliminate NH3 or Am to afford bifunctional lesions. This conclusion is further supported by nuclear magnetic resonance spectroscopic and enzymatic digestion analyses of the products of the reactions of these triamine complexes with d(GpG) and dG, which also reveal monofunctional binding. When cis-[Pt(NH3)2(4-Br-pyridine)Cl]+ was allowed to stand in phosphate-buffered saline at 37 degrees C for 14 days, however, NH4+ was released and trans-[Pt(NH3)(4-Br-pyridine)Cl2] formed concomitantly. This compound was characterized by a single crystal X-ray diffraction study, the details of which are reported. The fact that trans-[Pt(NH3)(4-Br-pyridine)Cl2] displays no anticancer activity, however, indicates that its formation from cis-[Pt(NH3)2(4-Br-pyridine)Cl]+ is not a significant component of the mechanism of action of this platinum-triamine complex. Taken together, these findings indicate that the cytotoxicity of cis-[Pt(NH3)2(Am)Cl]+ complexes most likely arises from the formation of monofunctional adducts. The DNA binding properties associated with this new class of antitumor agents suggest that they may display an activity profile different from that of cisplatin and related analogues.
Asunto(s)
Cisplatino/farmacología , Replicación del ADN/efectos de los fármacos , ADN/metabolismo , Cisplatino/análogos & derivados , Cisplatino/química , Cisplatino/metabolismo , ADN/química , Relación Estructura-ActividadRESUMEN
An SV40-based in vitro replication system has been used to examine the effects of platinum compounds on eukaryotic DNA replication. Plasmid templates containing the SV40 origin of replication were modified with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) or the inactive analogues [Pt(dien)Cl]+ and trans-DDP. The platinated plasmids were used as templates for DNA synthesis by the DNA polymerases present in cytosolic extracts prepared from human cell lines HeLa and 293. Bifunctional adducts formed by cis- and trans-DDP inhibited DNA replication by 95% at a bound drug to nucleotide ratio [(D/N)b] of less than 9 x 10(-4), in contrast to the monofunctional [Pt(dien)Cl]+ analogues, which required a (D/N)b of 3.4 x 10(-3) for 62% inhibition of DNA replication. An average of two platinum adducts per genome was sufficient for inhibition of DNA replication by cisplatin. When trans-DDP-modified, but not cis-DDP-modified, SV40 origin containing plasmids [(D/N)b = 1.7 x 10(-3)] were allowed to incubate in the 293 cytosolic extracts for 1 h prior to addition of T-antigen to initiate replication, DNA synthesis was restored to 30% of control. This result suggested the presence of an activity in the extracts that reactivates trans-DDP-modified DNA templates for replication. This hypothesis was confirmed by an in vitro nucleotide excision repair assay that revealed activity in 293 and HeLa cell extracts selective for trans-DDP-modified plasmid DNAs. Such selective repair of trans-DDP-damaged DNA in human cells would contribute to its lack of antitumor activity.