RESUMO
The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Fase G2/efeitos dos fármacos , Metilnitronitrosoguanidina/farmacologia , Neoplasias/genética , Alquilantes/farmacologia , Carcinoma , Neoplasias do Colo , Feminino , Humanos , Modelos Genéticos , Proteína 2 Homóloga a MutS , Neoplasias Ovarianas , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Deleção de Sequência , Células-Tronco , Células Tumorais CultivadasRESUMO
The human colon tumor cell line HCT 116 is known to have a homozygous mutation in the mismatch repair gene hMLH1 on human chromosome 3, to exhibit microsatellite instability, and to be defective in mismatch repair. In order to determine whether the introduction of a normal copy of hMLH1 gene restores mismatch repair activity and corrects microsatellite instability, a single human chromosome 3 from normal fibroblasts was transferred to HCT 116 cells via microcell fusion. As a control, human chromosome 2 was also transferred to HCT 116 cells. Two HCT 116 microcell hybrid clones that received a single copy of chromosome 2 (HCT 116 + ch2) and two that received a single copy of chromosome 3 (HCT 116 + ch3) were isolated and characterized. A G-G mismatch in M13-derived heteroduplex DNA was efficiently repaired in cell extracts from HCT 116 + ch3 cells, but not in those of parent HCT 116 cells or HCT 116 + ch2 cells. Microsatellite alterations at the D5S107 locus containing CA repeats were seen in 8 of 80 subclones from HCT 116 cells, and in 13 of 150 subclones from HCT 116 + ch2 cells. In contrast, none of the 225 subclones derived from mismatch repair-proficient HCT 116 + ch3 cells showed alterations in the microsatellite at the same locus. The effect of introducing chromosome 3 on the sensitivity of HCT 116 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined, since enhanced tolerance to MNNG is accompanied by loss of mismatch repair activity in several cell lines. Within 3 days after treatment with 5 microM MNNG, HCT 116 + ch3 cells became morphologically flat and stopped growing. Their colony-forming ability, determined 10 days after treatment, was reduced 200-fold when compared to MNNG-treated parental HCT 116 and HCT 116 + ch2 cells. These results support the hypothesis that mutations in both alleles of the hMLH1 gene are necessary for the manifestation of defective mismatch repair and microsatellite instability and for enhanced MNNG tolerance. The results also suggest that the mismatch repair system contributes to the process that causes growth arrest in response to DNA damage by alkylating agents.
Assuntos
Cromossomos Humanos Par 3/fisiologia , Neoplasias do Colo/genética , Reparo do DNA/genética , DNA Satélite/genética , Metilnitronitrosoguanidina/farmacologia , Mutação Puntual/genética , Sequência de Bases , Bandeamento Cromossômico , Cromossomos Humanos Par 2 , Tolerância a Medicamentos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Bannayan-Riley-Ruvalcaba syndrome (BRRS) is a rare hamartomatous polyposis condition with features of macrocephaly, intestinal juvenile polyposis, developmental delay, lipomas, and pigmentation spots of the male genitalia. An autosomal dominant pattern of inheritance exists in some families, but others appear as sporadic cases. Germ-line mutations in PTEN, a tyrosine phosphatase and putative tumor suppressor gene, have been demonstrated in two families with BRRS, and chromatin loss at the PTEN gene locus on chromosome 10q23 has been demonstrated in two BRRS patients. Germ-line mutations in PTEN have also been described in Cowden disease and in a small number of patients with juvenile polyposis syndrome. In an attempt to assess the nature of PTEN mutations in BRRS, we analyzed three sporadic BRRS patients for chromosome 10q23 deletion or PTEN germ-line mutations. All 3 patients demonstrated no loss of parental alleles at 15 chromosome 10q23 markers that encompassed the region of PTEN. In addition, analysis of mRNA and genomic DNA revealed no nonsense, missense, or insertion/deletion mutations of PTEN. Thus, other mechanisms besides mutation of PTEN must have occurred to cause BRRS in these patients. We speculate that BRRS and juvenile polyposis syndrome may have a heterogeneous etiology to cause their syndromes.
Assuntos
Mutação em Linhagem Germinativa/genética , Síndrome do Hamartoma Múltiplo/genética , Pólipos Intestinais/genética , Monoéster Fosfórico Hidrolases , Transtornos da Pigmentação/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Supressoras de Tumor , DNA/genética , Deficiências do Desenvolvimento/genética , Humanos , Masculino , PTEN Fosfo-Hidrolase , RNA Mensageiro/genética , SíndromeRESUMO
The hMSH2 protein plays an important role in the DNA mismatch repair system. Since this system is involved in the correction of errors that occur during DNA replication, we studied the expression of hMSH2 protein in resting and DNA-replicating cells, as well as through the cell cycle in cell types with different growth characteristics. Using Western blot analysis, we showed that hMSH2 protein was detectable in resting peripheral blood lymphocytes and thymocytes. However, when these cells were induced to proliferate, the protein level increased at least 12-fold. In cell-cycle dependent expression studies we chose two DNA mismatch repair proficient cell lines (HEL and HeLa-S3), and flow cytometry was used to monitor cell-cycle progression. At every phase in the cell cycle, the steady-state level of hMSH2 was higher than in resting lymphocytes or thymocytes, and only minor variations of expression level were observed through the cell cycle. In particular, a two to fourfold decrease in hMSH2 expression occurred at G1 in HEL and at early S phase in HeLa-S3, but higher expression levels resumed during the replicative and postreplicative phases of the cell cycle. Interestingly, hMSH2 protein expression decreased fourfold when HEL cells were induced to differentiate along the megakaryocyte lineage, when continuous DNA replication occurs without mitosis. These results suggest that a basal level of hMSH2 protein expression is necessary for resting and differentiated cells, and that increased hMSH2 protein expression is required when DNA replication is activated and followed by mitosis.
Assuntos
Ciclo Celular/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA , Proteínas Proto-Oncogênicas/metabolismo , Ciclo Celular/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Citometria de Fluxo , Fase G1/fisiologia , Células HeLa , Humanos , Interfase/fisiologia , Linfócitos/citologia , Linfócitos/metabolismo , Mitose/fisiologia , Proteína 2 Homóloga a MutSRESUMO
We have reported that transfer of chromosome 3 (Chr3) containing a single wild-type copy of the hMLH1 gene into HCT116 colon cancer cells, a cell line deficient in DNA mismatch repair (MMR) activity attributable to inactivating hMLH1 mutations, corrects all of the aspects of the MMR repair-deficient phenotype. We inhibited the expression of the wild-type hMLH1 gene using antisense RNA in HCT116+Chr3 cells to determine if this would result in reversion to the MMR-deficient phenotype. Despite profound inhibition of hMLH1 expression, DNA MMR activity and alkylation sensitivity were not impaired in the antisense-transfected HCT116+Chr3 cells. Additionally, arrest of the cell cycle at the G2 phase with alkylation damage occurs in these cells, a phenotype associated with MMR proficiency. These results indicate that even with a reduction in the expression of hMLH1 protein below the limits of detection by Western blotting, DNA MMR activity remained fully functional (by direct DNA MMR activity assay). We would speculate that hMLH1 is expressed in substantially greater abundance than would be minimally necessary for DNA MMR and that minor reductions in the expression of this protein would not be sufficient to permit DNA MMR dysfunction. Alternatively, Chr3 may contain a second hMLH1 homologue that might overlap with the function of hMLH1.
Assuntos
Pareamento Incorreto de Bases , Cromossomos Humanos Par 3/genética , Reparo do DNA , DNA/metabolismo , Proteínas de Neoplasias/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Proteínas de Transporte , Ciclo Celular/efeitos dos fármacos , Clonagem Molecular , Neoplasias do Colo/genética , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Fase G2 , Células HeLa , Humanos , Proteína 1 Homóloga a MutL , Proteínas Nucleares , Fenótipo , Plasmídeos/metabolismo , RNA/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Colorectal cancer is one of the leading causes of cancer deaths in the Western world. More than 56,000 newly diagnosed colorectal cancer patients die each year in the United States. Available therapies are either not effective or have unwanted side effects. Epidemiological data suggest that dietary manipulations play an important role in the prevention of many human cancers. Curcumin the yellow pigment in turmeric has been widely used for centuries in the Asian countries without any toxic effects. Epidemiological data also suggest that curcumin may be responsible for the lower rate of colorectal cancer in these countries. Curcumin is a naturally occurring powerful anti-inflammatory medicine. The anticancer properties of curcumin have been shown in cultured cells and animal studies. Curcumin inhibits lipooxygenase activity and is a specific inhibitor of cyclooxygenase-2 expression. Curcumin inhibits the initiation of carcinogenesis by inhibiting the cytochrome P-450 enzyme activity and increasing the levels of glutathione-S-transferase. Curcumin inhibits the promotion/progression stages of carcinogenesis. The anti-tumor effect of curcumin has been attributed in part to the arrest of cancer cells in S, G2/M cell cycle phase and induction of apoptosis. Curcumin inhibits the growth of DNA mismatch repair defective colon cancer cells. Therefore, curcumin may have value as a safe chemotherapeutic agent for the treatment of tumors exhibiting DNA mismatch repair deficient and microsatellite instable phenotype. Curcumin should be considered as a safe, non-toxic and easy to use chemotherapeutic agent for colorectal cancers arise in the setting of chromosomal instability as well as microsatellite instability.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Curcumina/uso terapêutico , Inibidores de Ciclo-Oxigenase/uso terapêutico , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Pareamento Incorreto de Bases , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/prevenção & controle , Curcumina/farmacocinética , Curcumina/farmacologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacocinética , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Isoenzimas/metabolismo , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Many genetic disorders are reported to cause irreversible damage to the fetus before birth. In utero gene therapy may be an effective tool for correction of genetic disorders by replacing defective gene with normal one. There are many reasons for moving forward with in utero gene therapy. The most important reason is to provide early intervention as to prevent or slow dysfunction and morbidity. This approach may prove to be advantageous in rapidly replicating fetal cells, and less sensitive to immune response to vector or transgene product due to underdeveloped immune system. In addition, the developing fetus may be a better candidate for gene therapy than the adult because gene engraftment may be more feasible in early fetal life, where stem cells or pleuripotent progenitor cells are more accessible to vectors. Some reports are available on successful in utero gene transfer in animal models but many questions remain to be answered before in utero gene therapy can be considered a viable solution to human. The real moral challenge facing in utero gene therapy is finding ways to insure that the review of protocols is adequate, and that those undertaking trials are competent to do so. Present review article analyzes the overall progress of the field, and the research that still needs to be performed before it can be considered to human clinical trials.
Assuntos
Doenças Fetais , Doenças Genéticas Inatas , Terapia Genética/tendências , Animais , Feminino , Doenças Fetais/embriologia , Doenças Fetais/genética , Doenças Fetais/terapia , Doenças Genéticas Inatas/embriologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Humanos , GravidezRESUMO
Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Cyclooxygenase (COX)-2 plays an important role in colon carcinogenesis. To investigate the effect of curcumin on COX-2 expression, we treated HT-29 human colon cancer cells with various concentrations of curcumin. Curcumin inhibited the cell growth of HT-29 cells in a concentration- and time-dependent manner. Curcumin markedly inhibited the mRNA and protein expression of COX-2, but not COX-1. These data suggest that a non-toxic concentration of curcumin has a significant effect on the in vitro growth of HT-29 cells, specifically inhibits COX-2 expression, and may have value as a safe chemopreventive agent for colon cancer.
Assuntos
Anticarcinógenos/farmacologia , Curcumina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Dieta , Células HT29 , Humanos , Isoenzimas/genética , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/análiseRESUMO
The effects of a number of phenobarbital-type inducers on selected drug-metabolizing enzymes in male F344/NCr rats were determined by measuring specific catalytic activities and/or by measuring the levels of RNA which hybridize with specific probes for the corresponding genes. The effects on hepatic CYP2B1 were assessed by measuring the levels of CYP2B1-specific RNA and benzyloxyresorufin O-dealkylase and testosterone 16 beta-hydroxylase activities. Levels of CYP3A were monitored by measuring the rate of hydroxylation of testosterone at the 6 beta-position. Microsomal epoxide hydrolase activity was determined by measurement of cellular RNA specific for this form and by assaying the hydrolysis of benzo[a]pyrene-4,5-oxide. UDP-glucuronyltransferase activity was assayed by measuring the glucuronidation of 3-hydroxybenz[a]anthracene. Levels of glutathione S-transferase Ya/Yc were measured by quantifying total cellular RNA coding for the proteins. When male F344/NCr rats were administered various doses of phenobarbital or dichlorodiphenyltrichloroethane (DDT), strong correlations between the induction of CYP2B1 and the induction of epoxide hydrolase or UDP-glucuronyltransferase activities were observed. Treatment of rats with barbiturates, hydantoins, halogenated pesticides such as DDT or alpha-hexachlorocyclohexane, 2,4,5,2',4',5'-hexachlorobiphenyl, CYP2B1 inhibitors such as clotrimazole or clonazepam, or such structurally-diverse compounds as 2-hexanone or diallyl sulfide resulted in induction of CYP2B1-mediated enzyme activity and induction of certain other forms of cytochrome P450, microsomal epoxide hydrolase, at least one form of UDP-glucuronyltransferase, and multiple forms of glutathione S-transferase. This suggests that, as a class, compounds which induce CYP2B1 also induce a coordinate hepatic pleiotropic response which includes induction of these other phase I and phase II drug-metabolizing enzymes.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/biossíntese , DDT/farmacologia , Glucuronosiltransferase/biossíntese , Glutationa Transferase/biossíntese , Fenobarbital/farmacologia , Animais , Sequência de Bases , Citocromo P-450 CYP1A1 , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática/efeitos dos fármacos , Epóxido Hidrolases/biossíntese , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Oxirredutases/biossíntese , RNA/biossíntese , Ratos , Ratos Endogâmicos F344 , Esteroide Hidroxilases/biossíntese , Xenobióticos/farmacologiaRESUMO
'Home made brew' (illicit liquor) is often consumed in rural areas of India. Forty-six such alcohol samples were collected from different areas to assess total acidity, pH and ethanol content. The mean ethanol content was 16.47% (v/v) (range 1.11-36.88%). Total acidity ranged between 0.56 and 10.88 with the mean of 2.85 g of sodium hydroxide/l. The mean pH was 4.20 (range 2.70-9.41).
Assuntos
Consumo de Bebidas Alcoólicas/etnologia , Bebidas Alcoólicas/análise , Etanol/análise , Humanos , Concentração de Íons de Hidrogênio , ÍndiaRESUMO
Erythrocyte superoxide dismutase (SOD: EC 1.15.1.1), catalase (EC 1.15.1.6), glucose-6-phosphate dehydrogenase (G-6-PD: EC 1.1.1.49), reduced glutathione (GSH) and malonyldialdehyde (MDA) were studied in 27 patients with chronic and 11 patients with acute renal failure. A comparison with 15 age- and sex-matched healthy subjects showed that patients with both acute and chronic renal failure had significantly low G-6-PD (p less than 0.05) values whereas SOD, catalase and MDA showed significantly elevated levels (p less than 0.05). After adequate dialysis or renal transplantation the SOD, G-6-PD, catalase and MDA values returned to normal. The findings suggest that the erythrocyte SOD, catalase, and G-6-PD can undergo an adaptive alteration which however appears reversible.
Assuntos
Catalase/sangue , Glucosefosfato Desidrogenase/sangue , Glutationa/sangue , Malonatos/sangue , Malondialdeído/sangue , Superóxido Dismutase/sangue , Uremia/sangue , Injúria Renal Aguda/sangue , Eritrócitos/metabolismo , Humanos , Falência Renal Crônica/sangue , Transplante de Rim , Diálise Renal , Uremia/terapiaRESUMO
The toxicological literature is replete with studies attempting to explain the mechanism of action of organophosphorus (OP) insecticides to their anticholinesterase activities, but not much is known about the metabolism and detoxification of these compounds. The goal of this study was to ascertain the toxic effects of chlorpyrifos, one of the most widely used OPs, on the liver of male rats and also to evaluate the protective potential of zinc in mediating its toxic effects. It was observed that chlorpyrifos (13.5 mg/kg body weight) treatment resulted in significant inhibition (p < 0.001) of serum and hepatic acetylcholinesterase (AChE) activities after 8 wk. However, zinc-treated (227 mg/L drinking water) animals resulted in significant normalization of the inhibited AChE activities. Similarly, a significant increase in the levels of various serum and liver marker enzymes (viz. alkaline phosphatase, aspartate aminotransferase [AST], and alanine aminotransferase [ALT]) was observed following treatment with chlorpyrifos. However, coadministration of zinc to these animals restored these enzymes to within normal limits, even though some increase in the activity of serum ALT and hepatic alkaline phosphatase still persisted at the end of the study. Chlorpyrifos treatment diminished serum and hepatic zinc levels significantly (p < 0.01 to p < 0.001) compared to normal control animals. Serum iron concentrations also plummeted significantly following chlorpyrifos treatment. On the contrary, serum copper levels were significantly increased (p < 0.01) in chlorpyrifos-treated animals, but they were normalized following zinc supplementation to the rats in this group. Interestingly, chlorpyrifos treatment resulted in elevated hepatic levels of copper, iron, and selenium, but zinc treatment could only partially restore the raised elemental concentrations. These data clearly demonstrate the potential role of zinc in mediating the toxic effects of chlorpyrifos, presumably because of their antioxidant properties and also their possible interaction with other trace elements in maintaining the cellular harmony.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Clorpirifos/intoxicação , Inseticidas/intoxicação , Oligoelementos/metabolismo , Zinco/uso terapêutico , Animais , Peso Corporal/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Clorpirifos/sangue , Enzimas/sangue , Inseticidas/sangue , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espectrometria por Raios X , Zinco/sangueAssuntos
Membrana Eritrocítica/enzimologia , Malária/enzimologia , Oxirredutases/sangue , Animais , Catalase/sangue , Glucosefosfato Desidrogenase/sangue , Glutationa/metabolismo , Glutationa Peroxidase/sangue , Glutationa Redutase/sangue , Masculino , Camundongos , Oxirredução , Plasmodium berghei , Superóxido Dismutase/sangueRESUMO
Literature on fetal metabolism of carcinogens since 1980 has been systematically listed and selectively discussed. The published data continues to support the conclusion that animal and human fetal tissues have the capacity to metabolize carcinogens, but at a low rate compared to the adult. Metabolism of low-molecular-weight chemicals, including nitrosamines, appears only near term in the rodent and is poorly inducible transplacentally; these agents are correspondingly relatively ineffective as fetal carcinogens. Metabolism of aromatic carcinogens, by contrast, appears early in gestation and is highly inducible transplacentally in rodents by chemicals such as polycyclic aromatic hydrocarbons (PAH) and tetrachlorodibenzo-p-dioxin, resulting in dramatic percent increases in enzyme activity. Transplacental induction has not been unequivocally demonstrated for the human fetus. Phase II enzymes, metabolizing aromatic compounds to water-soluble forms, generally have higher constitutive activity but lower degree of inducibility in the fetus, compared with phase I (activating) enzymes, and appear to show quantitatively different patterns in the human compared with the rodent. Specific and sensitive new technologies, including 32P-postlabelling, immunodetection of specific proteins, and use of cDNA probes, are beginning to be applied to fetal systems and are providing a more definitive and detailed understanding of the ontogeny and modulation of fetal carcinogen metabolizing enzymes. Fetal and maternal metabolism of PAH, especially methylcholanthrene (MC), have been found to be important determinants in susceptibility of the fetus to tumorigenesis. In particular, we have utilized a mouse model system wherein a single dominant gene, Ah, confers responsiveness to induction of PAH metabolism by cytochrome Pl450 (IA1); the recessive allele, Ah, is associated with nonresponsiveness. In appropriate backcrosses between C57BL/6 (AhAh) and DBA/2 (AhAh) mice, responsive and nonresponsive fetuses were carried together in mothers who were, themselves, either responsive or nonresponsive. In both cases, responsive fetuses later developed more lung and liver tumours after transplacental MC, compared with nonresponsive littermates. Fetuses of responsive mothers, however, experienced a lower cancer risk than did those of nonresponsive mothers at a comparable MC dose. Pretreatment of the pregnant mice with the noncarcinogenic inducer, beta-naphthoflavone (BNF) had a uniform protective effect for all of the fetuses, especially the responsive ones, if the mother was responsive. For nonresponsive mothers, by contrast, BNF pretreatment led to an enhancement of tumorigenesis, in the responsive fetuses only, under certain conditions of dose and fetal sex.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Carcinógenos/metabolismo , Troca Materno-Fetal , Animais , Carcinógenos/farmacologia , Indução Enzimática , Feminino , Expressão Gênica/efeitos dos fármacos , Isoenzimas/biossíntese , Compostos Policíclicos/metabolismo , GravidezRESUMO
Treatment of pregnant mice with 3-methylcholanthrene (MC) causes lung and liver tumors in the offspring, the incidences of which are greatly influenced by the Ah locus regulated induction phenotype for aryl hydrocarbon hydroxylase activity (AHH) in both the mother and fetuses. In order to examine the biochemical and molecular mechanisms responsible for the modulating effect of maternal environment on tumor susceptibility, reciprocal crosses between responsive C57BL/6 and non-responsive DBA/2 mice were made and the pregnant mothers were treated i.p. on the 17th day of gestation with either olive oil alone, 30 mg/kg of MC, or 30 mg/kg of beta-naphthoflavone (beta NF). At various times after injection, the mothers were killed and the fetuses removed for enzymatic and molecular blot analysis. In fetal lung tissues, the absolute levels and relative induction ratios of AHH activity from D2B6F1 fetuses were very similar to those obtained in B6D2F1 fetuses during the first 24 h following a transplacental exposure to either inducing agent. This was also the case 48 h after an injection of beta NF. However, 48 h after exposure to MC, the AHH activity in fetal lungs from B6 mothers had declined to practically control values, whereas fetal lungs from D2 mothers still exhibited a high level of AHH activity. Similar induction kinetics for the CYPIA1 gene were obtained in fetal livers. These results were confirmed at the RNA level by quantitative slot-blot analysis of fetal RNA preparations. In both organs, treatment with inducing agents for the P450IA1 gene resulted in a rapid and early induction of CYPIA1 RNA by 4 h. Fetuses from D2 mothers, however, showed a more sustained induction of CYPIA1 RNA following exposure to MC than did fetuses from B6 mothers. These results suggest that the observed increase in tumor susceptibility observed in the offspring of D2 mothers compared to the offspring of B6 mothers was due, at least in part, to the differences in the persistence of induction of the CYPIA1 gene locus, and may be the result of differences in the clearance rates of MC from the fetal and maternal compartments or its pharmacokinetic distribution in the two types of maternal environments.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Fígado/enzimologia , Pulmão/enzimologia , Metilcolantreno/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Cruzamentos Genéticos , Sistema Enzimático do Citocromo P-450/genética , Suscetibilidade a Doenças , Indução Enzimática/genética , Feminino , Feto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Troca Materno-Fetal , Camundongos , Mães , Fenótipo , Gravidez , RNA/análiseRESUMO
The ontogeny and transplacental inducibility of UDP-glucuronosyl transferase (UDPGT) activities potentially relevant to detoxification of polycyclic aromatic hydrocarbons were studied in (C57BL/6 x DBA/2) F1 or (DBA/2 x C57BL/6) F1 fetal mouse liver, with p-nitrophenol (PNP) and 3-hydroxybenzo[a]pyrene (3-OH-BP) as substrates. Both UDPGT activities developed during the late fetal period and reached almost 60% of the adult activity at term; PNP, but not 3-OH-BP UDPGT decreased significantly on postnatal day 1 before rising to adult levels. A single exposure to beta-naphthoflavone (beta NF; 150 mg/kg) on day 17 of gestation induced the PNP-UDPGT activity significantly (1.5-fold) by day 19 in the B6D2 F1s but not D2B6 F1s. A single dose of 3-methylcholanthrene (MC; 100 mg/kg) or 2,3,7,8,-tetrachlorodibenzo-p-dioxin (10 micrograms/kg) did not induce, but three injections of MC also resulted in significant induction in the fetuses of C57BL/6 mothers. 3-OH-BP-UDPGT was not significantly induced by any of the chemicals in either genetic cross. In a parallel study, a gene for an inducible mouse UDPGT, designated UDPGTm-1, was shown by Northern blotting to be expressed in fetal liver by day 18 of gestation at low levels relative to adults, but was not induced transplacentally by MC, beta NF or phenobarbital (PB). These results show that (1) at least two functionally defined UDPGT activities toward phenolic substrates are present in the late fetal mouse liver; (2) one of these is transplacentally inducible by beta NF and MC, but only in fetuses of C57BL/6 mothers, (3) induction where achieved was relatively small in magnitude, and (4) a gene of a PB-inducible UDPGT was expressed at low levels in the fetuses but was not induced transplacentally.
Assuntos
Feto/enzimologia , Glucuronosiltransferase/biossíntese , Fígado/enzimologia , Animais , Benzoflavonas/farmacologia , Indução Enzimática , Expressão Gênica , Idade Gestacional , Fígado/embriologia , Metilcolantreno/farmacologia , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Camundongos Endogâmicos DBA/embriologia , Camundongos Endogâmicos/embriologia , Dibenzodioxinas Policloradas/farmacologia , beta-NaftoflavonaRESUMO
Previous studies have shown that the incidences of liver and lung tumors in mice exposed transplacentally to 3-methyl-cholanthrene (MC) were significantly influenced by the sensitivity of both mothers and fetuses to induction of cytochrome(s) P-450 by polycyclic aromatic hydrocarbons. In order to delineate further the biochemical and molecular processes underlying the observed biological effects, the inductive effect of MC and beta-naphthoflavone (beta NF) on cytochrome P-450 was determined at the biochemical and molecular levels. C57BL/6 females were mated with DBA/2 males and treated i.p. on day 17 of gestation with olive oil alone, 150 mg/kg of beta NF or different doses of MC. At various times after injection the mothers were sacrificed and the fetuses removed for biochemical and molecular studies. MC caused maximal induction of aryl hydrocarbon hydroxylase (AHH) activity by 8 h in both the liver and lung. beta NF caused nearly maximal induction of AHH activity by 8 h in the lung but had little effect on liver AHH activity at this time. Maximal induction with beta NF occurred by 24 h in both organs. Addition of monoclonal antibody 1-7-1, specific for the MC-inducible forms of cytochrome P-450 (P-450IA1 and A2), to the incubation mixtures resulted in a 55-70% inhibition of AHH activity in both lung and liver assays, regardless of the inducing agent used, while having no effect on AHH activity from oil-treated mice. RNA blot analysis carried out in parallel with enzyme assays demonstrated that the levels of enzyme activity correlated very well with the levels of steady-state RNAs. MC caused maximal induction of P-450IA1 RNA levels 4 h after injection in both organs and a biphasic secondary increase was observed in the lung. Maximal levels of P-450IA1 RNA were seen at 12-16 h following injection of beta NF. However, the ratio of P-450IA1 RNAs present at 16 versus 2 h in the beta NF-treated liver appeared greater than that in the lung. P-450IA2 was also induced in fetal liver and lung, but at low levels relative to P-450IA1. The results indicate that the increase in functional AHH activity was primarily due to induction of cytochrome P-450IA1. The differences in induction kinetics observed for cytochromes P-450IA1 and A2 suggest that these enzymes exhibit both tissue- and inducer-dependent specificity.
Assuntos
Benzoflavonas/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Flavonoides/farmacologia , Fígado/enzimologia , Pulmão/enzimologia , Metilcolantreno/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática , Feminino , Feto , Fígado/efeitos dos fármacos , Fígado/embriologia , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Gravidez , RNA/efeitos dos fármacos , RNA/genética , Valores de Referência , beta-NaftoflavonaRESUMO
Pregnant Sprague-Dawley rats were treated with a single ip dose of either olive oil or 40 mg/kg of 3-methylcholanthrene on gestation day 20 and sacrificed at various times after injection. Determination of aryl hydrocarbon hydroxylase activity 24 hr after injection revealed that treatment with 3-methylcholanthrene resulted in a 10.5-fold stimulation of enzymatic activity in liver 800 x g supernatants. Western blot analysis with monoclonal antibody 1-7-1 confirmed these results and demonstrated the presence of a 3-methylcholanthrene-inducible P-450 isozyme. Using Northern and slot blot techniques, the induction of steady-state levels of CYPIA1 RNA was shown to occur as early as 4 hr following 3-methylcholanthrene injection. CYPIA1 RNA levels were induced 31.6-fold over values obtained from oil-treated tissues at this time. This appears to be the optimal time to study changes in the levels of CYPIA1 RNA gene expression in the fetus following transplacental exposure to polycyclic aromatic hydrocarbons. By 12 to 24 hr postinjection, the induction of CYPIA1 RNA levels declined to 3.5- to 8.5-fold above control values. These results demonstrate that the kinetics of induction of the CYPIA1 gene during the fetal period differed from that seen in adults.