RESUMEN
BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) constitute a large group of compounds that are water, stain, and oil repellent. Numerous sources contribute to the blood levels of PFAS in the European population. The main contributor for perfluorooctanoic acid (PFOA) is food, house dust, consumer products and personal care products (PCPs). OBJECTIVES: The purpose of the present work is to calculate the dietary and dermal external exposure to PFOA, estimate the aggregated internal exposure from diet and PCPs using a PBPK model, and compare estimates with measured concentrations. METHODS: Detailed information on diet and PCP use from the EuroMix study is combined with concentration data of PFOA in food and PCPs in a probabilistic exposure assessment. A physiologically based pharmacokinetic model (PBPK) was further refined by incorporating a dermal exposure pathway, and changes in the kidney and faecal excretion. RESULTS: The aggregated internal exposure using the PBPK model shows that the major contributor to the internal exposure is diet for both males and females. The estimated internal exposure of PFOA for the EuroMix population was in the same range but lower than the measured blood concentrations using the lower bound (LB) external exposure estimates, showing that the LB estimates are underestimations. For seven females the internal exposure of PFOA were higher from PCPs than from diet. CONCLUSION: PCPs and diet contributed in the same range to the internal PFOA exposure for several women participating in EuroMix. This calls for additional studies on exposure to PFOA and possibly other PFAS from PCPs, especially for women. Overall, PBPK modelling was shown as valuable tool in understanding the sources of PFOA exposure and in guiding risk assessments and regulatory decisions.
Asunto(s)
Ácidos Alcanesulfónicos , Cosméticos , Contaminantes Ambientales , Fluorocarburos , Masculino , Humanos , Femenino , Monitoreo Biológico , Dieta , Noruega , Contaminantes Ambientales/metabolismoRESUMEN
Phthalates are diesters of phthalic acid and have been widely used as plasticizers in polyvinyl chloride (PVC) plastics. Phthalates are also used as excipients in pharmaceuticals and personal care products (PCPs). Phthalates can migrate from the plastic into the air, water and food, and humans can be exposed via multiple pathways such as dermal, oral and inhalation. There is evidence that phthalates can induce reproductive and developmental toxicity not only in experimental animals but also in humans through disruption of estrogenic activity. The aim of this study was to collect concentration data on five phthalates in foods and PCPs from the scientific literature and combine these with food consumption data and PCP use frequency data from the EuroMix biomonitoring (BM) study in order to assess exposure. Probabilistic exposure assessments of phthalates were performed from foods and PCPs. Due to the very limited data available in the literature for DINCH, an exposure assessment was not carried out for this compound. The food groups with the highest contribution to phthalates exposure were: beverages, dairy, bread and meat products. The exposure estimates were compared with the measured phthalate metabolite levels from 24-hour urine samples. Regarding the oral route, measured phthalate exposure was between the lower bound (LB) and medium bound (MB) estimated exposure for all phthalates, except for DEP. The measured exposure from urine correlated with the estimated exposure from food for DEHP and DBP, while for BBP and DEP it correlated with the exposure estimates from PCPs. There were no significant differences between the BM data and the estimated exposure, except for DINP for males (p = 0.01). The LB and MB phthalate exposures estimated from foods and PCPs and the measured exposure from the urine were considerably lower than their respective tolerable daily intake (TDI) values established by the European Food Safety Authority (EFSA) and the World Health Organization (WHO). For the upper bound (UB), the exposure estimates are approximately double the TDI; however, this is regarded as a worst-case estimate and has low correlation with the measured exposure.
Asunto(s)
Cosméticos , Ácidos Ftálicos , Animales , Monitoreo Biológico , Exposición a Riesgos Ambientales/análisis , Humanos , MasculinoRESUMEN
BACKGROUND: Human exposure to chemicals through the oral, dermal, or inhalation routes is significant. To assess this exposure, a human biomonitoring study was conducted in Norway to examine the plausibility of source-to-dose calculations for chemical mixtures. Per- and polyfluoroalkyl substances (PFASs) are man-made compounds used for their surfactant properties, and several are persistent and bioaccumulative. Some PFASs are toxic and are regarded as endocrine disruptors and have been shown to suppress immune function and affect cholesterol homeostasis. Using the participants from the EuroMix BM study, we set out to describe PFAS concentrations and to evaluate associations with diet and use of personal care products (PCPs). METHODS: Participants (44 males and 100 females) kept detailed diaries on their food consumption and their PCP use for two non-consecutive days. All urine (24 h) and blood samples were collected at the end of each study day. Levels of 25 PFASs were analysed in serum from study day 1 using a high throughput online solid phase extraction ultra-high-performance liquid chromatography tandem mass spectrometry method. Multivariable linear regressions were performed between each food and PCP category and each chemical and were sex-stratified when the consumption of food or use of PCPs was significantly different between men and women. RESULTS: Eight PFASs were detected in all analysed samples (PFHxS, PFHpS, PFOS, PFOA, PFNA, PFDA, PFUnDA and PFDoDA), and four PFASs were below the limit of detection (PFOPA, PFDPA, PFHxA, and EtFOSA). Several PFASs were found to be positively associated with fish consumption (PFOS, PFNA, PFUnDA, PFDoDA, PFDA, PFDS and PFTrDA). Sunscreen, mouthwash, and lip gloss/lip balm were found to be positively associated with PFASs (PFOA, PFTrDA, and PFOSA). CONCLUSION: The participants in the EuroMix study were exposed to PFASs through their diet and PCP use. Several foods and PCPs were found to be potential sources of exposure to PFASs.
Asunto(s)
Ácidos Alcanesulfónicos , Cosméticos , Disruptores Endocrinos , Contaminantes Ambientales , Fluorocarburos , Ácidos Alcanesulfónicos/análisis , Animales , Monitoreo Biológico , Contaminantes Ambientales/análisis , Femenino , Fluorocarburos/análisis , Humanos , Masculino , NoruegaRESUMEN
Phthalates are widely used as plasticisers in flexible plastics and containers for food and personal care products (PCPs) and contaminates foods and PCPs. A human biomonitoring (BM) study was performed to study exposure of chemicals from foods and PCPs. For two 24-h periods, adult volunteers (n = 144) in Norway kept diaries on food eaten and usage of PCPs, and collected 24-h urine. Aggregated exposure to di(2-ethylhexyl) phthalate (DEHP) from dietary and PCPs was estimated by Monte-Carlo simulation using Oracle Crystal Ball©. Simulated urinary concentrations using physiologically based pharmacokinetic (PBPK) models were compared with measured urinary metabolites of DEHP, mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono-2-ethyl 5-carboxypentyl phthalate (MECCP). DEHP exposure from food are approximately 10 times higher than exposure than from PCPs. The main contributors to dietary exposure are dairy, grain, fruits and vegetables, meat and fish. Body lotion contribute most to the exposure of DEHP from PCPs. Forward-dosimetry gives good convergence with 24-h urinary concentrations of simulated and measured BM data. The measured concentration of the MECCP metabolite correlated well with simulated high exposure, while the measured concentrations of MEHP, MEHHP and MEOHP partly overlapped with both simulated low, medium and high metabolite exposure.
Asunto(s)
Monitoreo Biológico , Cosméticos/química , Dietilhexil Ftalato/administración & dosificación , Dietilhexil Ftalato/orina , Modelos Biológicos , Adulto , Anciano , Dieta , Dietilhexil Ftalato/metabolismo , Dietilhexil Ftalato/farmacocinética , Femenino , Contaminación de Alimentos , Humanos , Masculino , Persona de Mediana Edad , Estructura Molecular , Noruega , Adulto JovenRESUMEN
BACKGROUND: Exposure to multiple chemicals occurs daily through several routes; diet, inhalation and dermal contact. Real-life exposure assessment is needed to understand the risk. Therefore, a human biomonitoring (BM) study was performed to examine the plausibility of source-to-dose calculations for chemical mixtures in the Horizon 2020 EuroMix project. OBJECTIVES: To provide a detailed description of the design of the EuroMix BM study, and to present the initial results for urinary phenols and phthalates and to describe their exposure determinants from foods and personal care products (PCPs). METHOD: Adults (44 males and 100 females) kept detailed diaries on their food consumption, PCP use and handling of cash receipts. Urine samples were collected over the same 24-hour period. Urinary levels of four parabens, five bisphenols, oxybenzone/benzophenone-3 (OXBE), triclosan (TCS), triclocarban (TCC) and metabolites of eight phthalates and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) were analysed by ultra-high-performance liquid chromatography and tandem mass spectrometry. Multivariable linear regressions were performed between PCPs/food categories and each dependent chemical variable separately, and were only sex-stratified when an interactions between sex and the independent variable was significant. RESULTS: The detection rate for the metabolites of phthalates and DINCH, and bisphenol A (BPA) and TCS in urine was 88-100%, while bisphenol S (BPS) and bisphenol F (BPF) were only found in 29% and 4% of the urine samples, respectively. Bisphenol B (BPB), bisphenol AF (BPAF) and TCC were not detected. Food groups associated with phenol exposure were meat, bread, beverages and butter and oil. Food determinants for phthalate exposure were sweets, butter and oil, fruit and berries and other foods. The only positive association between the use of PCPs and phenols was found between BPA and lip gloss/balm. Phthalate exposure was associated with the use of shower gel, hand cream (females), toothpaste, anti-wrinkle cream (females) and shaving products (males). CONCLUSION: The participants in the EuroMix BM study were exposed to a mixture of phenols and phthalates. A variety of food categories and PCPs were found to be possible sources of these chemicals. This indicates a complex pattern of exposure to numerous chemicals from multiple sources, depending on individual diet and PCP preferences.
Asunto(s)
Contaminantes Ambientales/orina , Fenoles/orina , Ácidos Ftálicos/orina , Adulto , Anciano , Monitoreo Biológico , Cosméticos , Exposición Dietética , Femenino , Contaminación de Alimentos , Humanos , Masculino , Persona de Mediana Edad , Noruega , Adulto JovenRESUMEN
In the most recent risk assessment for Bisphenol A for the first time a multi-route aggregate exposure assessment was conducted by the European Food Safety Authority. This assessment includes exposure via dietary sources, and also contributions of the most important non-dietary sources. Both average and high aggregate exposure were calculated by source-to-dose modeling (forward calculation) for different age groups and compared with estimates based on urinary biomonitoring data (backward calculation). The aggregate exposure estimates obtained by forward and backward modeling are in the same order of magnitude, with forward modeling yielding higher estimates associated with larger uncertainty. Yet, only forward modeling can indicate the relative contribution of different sources. Dietary exposure, especially via canned food, appears to be the most important exposure source and, based on the central aggregate exposure estimates, contributes around 90% to internal exposure to total (conjugated plus unconjugated) BPA. Dermal exposure via thermal paper and to a lesser extent via cosmetic products may contribute around 10% for some age groups. The uncertainty around these estimates is considerable, but since after dermal absorption a first-pass metabolism of BPA by conjugation is lacking, dermal sources may be of equal or even higher toxicological relevance than dietary sources.
Asunto(s)
Compuestos de Bencidrilo , Exposición a Riesgos Ambientales/análisis , Contaminantes Ambientales , Fenoles , Adolescente , Adulto , Anciano , Niño , Preescolar , Dieta , Europa (Continente) , Femenino , Contaminación de Alimentos , Agencias Gubernamentales , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Modelos Teóricos , Papel , Absorción Cutánea , Adulto JovenRESUMEN
Coumarin is a naturally occurring flavouring substance in cinnamon and many other plants. It is known that coumarin can cause liver toxicity in several species, and it is considered a non-genotoxic carcinogen in rodents. By using the bench mark dose approach we re-assessed coumarin toxicity and established a new TDI for coumarin of 0.07 mg/kg bw/day. Oral intake of coumarin is related to consumption of cinnamon-containing foods and food supplements. Cinnamon is a widely used spice in Norway, and can be used as topping on oatmeal porridge. Based on analyses of coumarin in Norwegian foods, intake calculations for children and adults were conducted, and a risk assessment of coumarin in the Norwegian population was performed. Intake estimates of coumarin show that small children eating oatmeal porridge several times a week sprinkled with cinnamon could have a coumarin intake of 1.63 mg/kg bw/day and may exceeding the TDI with several folds. Adults drinking cinnamon-based tea and consuming cinnamon supplements also can exceed TDI. The coumarin intake could exceed the TDI by 7- to 20-fold in some intake scenarios. Such large daily exceedances of TDI, even for a limited time period of 1-2 weeks, cause concern of adverse health effects.
Asunto(s)
Avena/química , Cinnamomum zeylanicum/química , Cumarinas/efectos adversos , Exposición a Riesgos Ambientales , Medición de Riesgo , Adolescente , Adulto , Anciano , Niño , Cumarinas/análisis , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Noruega , Adulto JovenRESUMEN
5-Hydroxymethylfurfural (HMF) is formed in carbohydrate-rich food during acid-catalysed dehydration and in the Maillard reaction from reducing sugars. HMF is found in mg quantities per kg in various foods. HMF is mainly metabolised to 5-hydroxymethyl-2-furoic acid (HMFA), but unknown quantities of the mutagenic 5-sulphoxymethylfurfural (SMF) may also be formed, making HMF potentially hazardous to humans. We determined the HMF content in Norwegian food items and estimated the dietary intake of HMF in 53 volunteers by means of 24h dietary recall. The estimated intakes of HMF were correlated with urinary excretion of HMFA. Coffee, prunes, dark beer, canned peaches and raisins had the highest levels of HMF. The 95th percentile of the estimated daily dietary intake of HMF and the 24h urinary excretion of HMFA were 27.6 and 28.6mg, respectively. Coffee, dried fruit, honey and alcohol were identified as independent determinants of urinary HMFA excretion. Most participants had lower estimated HMF intake than the amount of HMFA excreted in urine. In spite of this there was a significant correlation (r=0.57, P<0.001) between the estimated HMF intake and urinary HMFA. Further studies are needed to reveal alternative sources for HMF exposure.
Asunto(s)
Furaldehído/análogos & derivados , Adulto , Cromatografía Líquida de Alta Presión , Dieta , Femenino , Análisis de los Alimentos , Furaldehído/toxicidad , Furaldehído/orina , Furanos/orina , Humanos , Masculino , NoruegaRESUMEN
A risk benefit assessment in Norway on the intake of added sugar, intense sweeteners and benzoic acid from beverages, and the influence of changing from sugar sweetened to diet beverages was performed. National dietary surveys were used in the exposure assessment, and the content of added sugar and food additives were calculated based on actual contents used in beverages and sales volumes provided by the manufactures. The daily intake of sugar, intense sweeteners and benzoic acid were estimated for children (1- to 13-years-old) and adults according to the current intake level and a substitution scenario where it was assumed that all consumed beverages contained intense sweeteners. The change from sugar sweetened to diet beverages reduced the total intake of added sugar for all age groups but especially for adolescent. This change did not result in intake of intense sweeteners from beverages above the respective ADIs. However, the intake of acesulfame K approached ADI for small children and the total intake of benzoic acid was increased to above ADI for most age groups. The highest intake of benzoic acid was observed for 1- to 2-year-old children, and benzoic acid intake in Norwegian children is therefore considered to be of special concern.
Asunto(s)
Bebidas/análisis , Carbohidratos/administración & dosificación , Carbohidratos/efectos adversos , Ingestión de Energía/efectos de los fármacos , Edulcorantes/efectos adversos , Edulcorantes/farmacología , Adolescente , Adulto , Factores de Edad , Anciano , Aspartame/efectos adversos , Aspartame/análisis , Ácido Benzoico/toxicidad , Bebidas/efectos adversos , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Noruega/epidemiología , Encuestas Nutricionales , Medición de Riesgo , Factores Sexuales , Tiazinas/efectos adversos , Tiazinas/análisisRESUMEN
Micronucleus (MN) induction in erythrocytes of multiple intestinal neoplasia (Min) mice with heterozygous Apc mutation was measured after s.c. injections of acrylamide, glycidamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and colchicine, and compared with wild-type (wt) mice. Since Apc influences microtubule dynamics, we wanted to test whether Min-mice were more sensitive to the production of MN than wild-type mice. We also examined the effect of pre-treatment with cytosine beta-D-arabinofuranoside (Ara C) and hydroxyurea, which inhibit ligation of DNA strand breaks in the repair of DNA adducts. All compounds induced a significant increase in MN in both strains of mice with the following potencies: acrylamideAsunto(s)
Acrilamida/toxicidad
, Proteína de la Poliposis Adenomatosa del Colon/genética
, Eritrocitos/efectos de los fármacos
, Imidazoles/toxicidad
, Micronúcleos con Defecto Cromosómico/inducido químicamente
, Mutágenos/toxicidad
, Poliposis Adenomatosa del Colon/genética
, Animales
, Colchicina/toxicidad
, Citarabina/farmacología
, Aductos de ADN/metabolismo
, Compuestos Epoxi/toxicidad
, Femenino
, Citometría de Flujo
, Hidroxiurea/farmacología
, Inyecciones Subcutáneas
, Masculino
, Ratones
, Ratones Endogámicos C57BL
, Ratones Mutantes
, Pruebas de Micronúcleos
RESUMEN
Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.
Asunto(s)
Uniones Comunicantes/efectos de los fármacos , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Carbazoles/farmacología , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Conexina 43/metabolismo , Relación Dosis-Respuesta a Droga , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-delta , Proteína Quinasa C-epsilonRESUMEN
The multiple intestinal neoplasia (Min)/+ mouse, which harbors only one functional allele of the Apc gene, is susceptible to environmental factors that disrupt this gene and subsequently trigger Apc-driven tumorigenesis in the colon. Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in colon carcinogenesis. Recently, we reported the absence of "classical" ACF in the colon of untreated Min/+ mice. Instead we identified flat dysplastic lesions, which we denoted ACF(Min) (J. E. Paulsen et al., Scand. J. Gastroenterol., 35: 534-539, 2000). In contrast to the classical type, ACF(Min) are not elevated above the surrounding mucosa, and their detection is totally dependent on methylene blue staining and transillumination. In the present study, we treated Min/+ mice with 5 mg/kg body weight azoxymethane (AOM) at weeks 1 and 2 and demonstrated induction of both types of lesions. However, only ACF(Min) appeared to be associated with the development of adenomas. Monocryptal ACF(Min), large ACF(Min), and adenomas showed a uniform histopathological picture of dysplasia and cytoplasmic overexpression of beta-catenin, indicating a qualitative relationship between these lesions. Also a quantitative relationship was suggested because the dramatic decrease in ACF(Min) number from week 7 to 11 was paralleled by a reciprocal increase in tumor number, indicating fast-crypt multiplication of ACF(Min). In AOM-treated +/+ (wild-type) littermates, a low number of ACF(Min) and tumors with the same characteristics as in Min/+ mice was seen. In contrast to ACF(Min), histopathological and immunohistochemical examination of classical ACF showed normal or hyperplastic crypts with normal levels of beta-catenin expression. In AOM-treated Min/+ mice, the number of classical ACF was virtually constant from week 7 to 11, and only a modest increase of crypt multiplicity was observed. The number of AOM-induced classical ACF at week 11 was not different in Min/+ mice and +/+ mice. In conclusion, we identified two distinct populations of altered crypts in the colon of Min/+ mice after AOM treatment. The ACF(Min), which resemble the dysplastic ACF described previously, clearly showed a continuous development from the monocryptal stage to adenoma, and they were characterized by fast-growing crypts with altered control of beta-catenin. In contrast, the classical ACF, which resemble the hyperplastic ACF described previously, were characterized by slow-growing crypts with normal beta-catenin expression, and they were probably not related to tumorigenesis.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias del Colon/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Transactivadores , Adenoma/genética , Adenoma/patología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Alelos , Animales , Azoximetano , Carcinógenos , División Celular/fisiología , Cocarcinogénesis , Neoplasias del Colon/inducido químicamente , Proteínas del Citoesqueleto/biosíntesis , Femenino , Genes APC , Predisposición Genética a la Enfermedad/genética , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Lesiones Precancerosas/inducido químicamente , beta CateninaRESUMEN
12-O:-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communication in many cell culture systems, but TPA-induced phosphorylation of the gap junction protein connexin43 (Cx43) varies much between systems. We have here studied whether these responses and their sensitivities can be correlated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved. Rat R6 fibroblasts transfected with the cDNA sequence encoding PKC beta I (R6-PKC3) had a total PKC activity 7- to 16-fold higher than the corresponding control cells (R6-C1), depending on the selection pressure (G418 concentration). Still, R6-PKC3 cells were no more sensitive than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 micrograms/ml G418). Thus, total PKC activity does not indicate absolute sensitivity of a cell system to TPA-induced suppression of communication, but within a certain cell system increasing PKC activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKC beta I is of minor importance for TPA-induced regulation of communication. Experiments with the Lilly compound 379196, a PKC beta-specific inhibitor, further supported this conclusion. Except for PKC beta I in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenzymes alpha, delta, epsilon and mu. Long-term treatment with TPA caused strong down-regulation of PKC alpha, delta and epsilon, but little down-regulation of PKC mu. Concurrently, the cells became refractory to repeated exposure to TPA, indicating that PKC mu is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKC alpha (and beta/gamma) inhibitor Gö6976 suggested that both classical (alpha) and novel PKCs (delta and epsilon) might be involved in TPA-induced suppression of intercellular communication, while phosphorylation of Cx43 may mainly be mediated by PKC alpha in the present systems.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Conexina 43/metabolismo , Fibroblastos/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Isoenzimas/fisiología , Proteína Quinasa C/fisiología , Acetato de Tetradecanoilforbol/farmacología , Animales , Western Blotting , Células Cultivadas , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibroblastos/enzimología , Isoenzimas/antagonistas & inhibidores , Ratones , Ratones Desnudos , Ésteres del Forbol/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C beta , RatasRESUMEN
The protein-tyrosine phosphatase inhibitors pervanadate, permolybdate, H2O2, and to a much lesser extent vanadate, increased the amount of cellular phosphotyrosine and induced tyrosine phosphorylation of connexin43 (Cx43) in early passage hamster embryo fibroblasts. The presence of phosphotyrosine in Cx43 immunoprecipitates from pervanadate-treated cells was shown by a phosphotyrosine-specific antibody and a phosphotyrosine-specific phosphatase. Pervanadate-induced Cx43 tyrosine phosphorylation was further verified by phosphoamino acid analysis, while no phosphotyrosine was present in control cells. This is the first observation of tyrosine phosphorylation of connexins in normal cells.
Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Fosfotirosina/metabolismo , Animales , Células Cultivadas , Cricetinae , Mesocricetus , Fosforilación , Vanadatos/metabolismoAsunto(s)
Comunicación Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Proteína Quinasa C/efectos de los fármacos , Animales , Bioensayo , Activación Enzimática , FosforilaciónRESUMEN
A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.
Asunto(s)
Comunicación Celular , Conexina 43/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Uniones Intercelulares/fisiología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Compuestos de Aluminio/farmacología , Animales , Comunicación Celular/efectos de los fármacos , Línea Celular , Conexina 43/aislamiento & purificación , Cricetinae , Cricetulus , Embrión de Mamíferos , Éteres Cíclicos/farmacología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Fluoruros/farmacología , Uniones Intercelulares/efectos de los fármacos , Radioisótopos de Yodo , Cinética , Pulmón , Toxinas Marinas , Mesocricetus , Ácido Ocadaico , Oxazoles/farmacología , Acetato de Tetradecanoilforbol/farmacología , Vanadatos/farmacología , Compuestos de Vanadio/farmacologíaRESUMEN
The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPP A), sapintoxin D (SAP D) and sapintoxin A (SAP A) on the decrease in [125I]epidermal growth factor (EGF) binding (indicating protein kinase C activation), suppression of gap junctional intercellular communication (GJIC) and induction of morphological cell transformation (MCT) in Syrian hamster embryo (SHE) cells were investigated. All five phorbol esters were found to reduce [125I]EGF binding in early passage SHE cells at comparable concentrations. DOPP A was approximately 10-fold less potent in decreasing GJIC compared to the other phorbol esters in early passage SHE cells, while the compounds showed less difference in suppressing GJIC in the phorbol ester sensitive SHE cell line BPNi. The decreases in [125I]EGF binding and GJIC were found to be transient in the continuous presence of phorbol esters. All phorbol esters induced MCT in early passage SHE cells, but DOPP and DOPP A were approximately 10-fold less potent than TPA, SAP D and SAP A. Thus, there seems to be some degree of correlation, but not to a full extent, between the ability of the phorbol esters to activate PKC, decrease GJIC and to induce MCT. The results do not suggest a simple relationship between PKC activation, inhibition of GJIC and the reported tumor-promoting activities of the compounds.
Asunto(s)
Transformación Celular Neoplásica , Factor de Crecimiento Epidérmico/efectos de los fármacos , Uniones Intercelulares/efectos de los fármacos , Ésteres del Forbol/toxicidad , Animales , Comunicación Celular/efectos de los fármacos , Línea Celular Transformada , Células Cultivadas , Cricetinae , Activación Enzimática/efectos de los fármacos , Mesocricetus , Proteína Quinasa C/metabolismoRESUMEN
Four different in vitro cytotoxicity tests were compared: the MTT assay, the NR assay, the uridine incorporation assay and the measurement of total cellular protein. The comparison was done using the BHK-21 cell line and nine selected test chemicals (colchicine, amitriptyline, cycloheximide, 2,5-hexandione, mercury chloride, cadmium chloride, copper chloride, 2,4-dinitrophenol and chloroquine diphosphate). The concentration that induced 50% inhibition relative to the controls (IC(50)) was calculated for each test and chemical. The results from the cytotoxicity tests were generally in good agreement. However, for some chemicals the IC(50) values varied significantly between two assays. The largest variation was found for chloroquine diphosphate, where the IC(50) value for the NR assay was approximately eight times lower than the IC(50) value for the protein measurement.