RESUMO
To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Intoxicação por Arsênico/prevenção & controle , Arsênio/metabolismo , ATPases Transportadoras de Arsenito/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Arsênio/farmacologia , Intoxicação por Arsênico/tratamento farmacológico , Arsenitos/metabolismo , Arsenitos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Células HEK293 , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Taxa de Sobrevida , Transfecção , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Arsenic resistance determinants from 42 environmental bacterial isolates (32 Gram negative) were analyzed by DNA: DNA hybridization using probes derived from Escherichia coli and Staphylococcus plasmid or chromosomal arsenic resistance (ars) genes. In colony hybridization assays, 11 and 1 Gram negative strains hybridized with the E. coli chromosome and plasmid probes, respectively. No hybridization was detected using a probe containing only the arsA (ATPase) gene from E. coli plasmid or with a Staphylococcus plasmid ars probe. From Southern hybridization tests of some of the positive strains it was concluded that homology to ars chromosomal genes occurred within chromosome regions, except in an E. coli isolate where hybridization occurred in both the chromosome and a 130-kb plasmid. Our results show that DNA sequences homologous to E. coli ars chromosomal genes are commonly present in the chromosomes of environmental arsenic-resistant Gram negative isolates.
Assuntos
Arsenicais/farmacologia , Bactérias/genética , Proteínas de Bactérias , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Bombas de Íon , Complexos Multienzimáticos , Fatores R/genética , Microbiologia do Solo , Adenosina Trifosfatases/genética , Arseniatos/farmacologia , ATPases Transportadoras de Arsenito , Arsenitos/farmacologia , Bactérias/efeitos dos fármacos , Cromossomos Bacterianos/genética , Escherichia coli/genética , México , Hibridização de Ácido Nucleico , Poluentes do Solo/farmacologia , Transativadores/genéticaRESUMO
Arsenic compounds, often present as environmental pollutants, are highly toxic for most microorganisms. Some microbial strains possess genetic determinants conferring resistance to arsenic derivatives. In bacteria, these determinants are usually located on plasmids, which has facilitated their analysis with molecular detail. Bacterial plasmids conferring arsenic resistance encode specific pumps that extrude arsenite (AsIII). In Gram-negative bacteria, the efflux pump consists of a complex formed by an ATPase (ArsA) associated with a membrane anion channel (ArsB). Arsenate (AsV) is converted to arsenite by a soluble reductase (ArsC). Proteins ArsB and ArsC, but not the ATPase, are also found in Gram-positive bacteria. Besides the widely spread plasmid arsenic resistance determinants, some bacteria possess the ability to enzimatically oxidize arsenite to less toxic arsenate.