Mitogen- and stress-activated protein kinases 1 and 2 are required for maximal trefoil factor 1 induction.

Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2), activated downstream of the ERK- and p38-mitogen-activated protein kinase pathways are involved in cell survival, proliferation and differentiation. Following mitogenic or stress stimuli, they mediate the nucleosomal response, which includes phosphorylation of histone H3 at serine 10 (H3S10ph) coupled with transcriptional activation of immediate-early genes. While MSK1 and MSK2 are closely related, their relative roles may vary with cellular context and/or stimuli. However, our knowledge of MSK2 recruitment to immediate-early genes is limited, as research has primarily focused on MSK1. Here, we demonstrate that both MSK1 and MSK2, regulate the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced expression of the breast cancer marker gene, trefoil factor 1 (TFF1), by phosphorylating H3S10 at its 5' regulatory regions. The MSK-mediated phosphorylation of H3S10 promotes the recruitment of 14-3-3 isoforms and BRG1, the ATPase subunit of the BAF/PBAF remodeling complex, to the enhancer and upstream promoter elements of TFF1. The recruited chromatin remodeling activity leads to the RNA polymerase II carboxy-terminal domain phosphorylation at the TFF1 promoter, initiating TFF1 expression in MCF-7 breast cancer cells. Moreover, we show that MSK1 or MSK2 is recruited to TFF1 regulatory regions, but as components of different multiprotein complexes.

Activation and function of immediate-early genes in the nervous system.

Immediate-early genes have important roles in processes such as brain development, learning, and responses to drug abuse. Further, immediate-early genes play an essential role in cellular responses that contribute to long-term neuronal plasticity. Neuronal plasticity is a characteristic of the nervous system that is not limited to the first stages of brain development but persists in adulthood and seems to be an inherent feature of everyday brain function. The plasticity refers to the neuron's capability of showing short- or long-lasting phenotypic changes in response to different stimuli and cellular scenarios. In this review, we focus on the immediate-early genes encoding transcription factors (AP-1 and Egr) that are relevant for neuronal responses. Our current understanding of the mechanisms involved in the induction of the immediate-early genes is presented.

Role of MSK1 in the malignant phenotype of Ras-transformed mouse fibroblasts.

Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth.

Current understanding and importance of histone phosphorylation in regulating chromatin biology.

The core histones H2A, H2B, H3 and H4, undergo various post-translational modifications, such as acetylation, methylation and phosphorylation. Core histone phosphorylation has roles in several biological responses, including transcription, mitosis, DNA repair and apoptosis. Histone phosphorylation may disrupt chromatin structure and/or provide a 'code' for the recruitment or occlusion of non-histone chromosomal proteins to chromatin. Among the better-characterized histone phosphorylation events are the phosphorylation of H3 at Ser10 and Ser 28, and the phosphorylation of the H2A variant H2A.X at Ser139. Much remains to be learned about the function of the many other core histone phosphorylation events in chromatin. This review provides an overview of the biological roles of core histone phosphorylation in mammalian cells.

Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex.

Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-kappaB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription.

H3 phosphorylation: dual role in mitosis and interphase.

Chromatin condensation and subsequent decondensation are processes required for proper execution of various cellular events. During mitosis, chromatin compaction is at its highest, whereas relaxation of chromatin is necessary for DNA replication, repair, recombination, and gene transcription. Since histone proteins are directly complexed with DNA in the form of a nucleosome, great emphasis is put on deciphering histone post-translational modifications that control the chromatin condensation state. Histone H3 phosphorylation is a mark present in mitosis, where chromatin condensation is necessary, and in transcriptional activation of genes, when chromatin needs to be decondensed. There are four characterized phospho residues within the H3 N-terminal tail during mitosis: Thr3, Ser10, Thr11, and Ser28. Interestingly, H3 phosphorylated at Ser10, Thr11, and Ser28 has been observed on genomic regions of transcriptionally active genes. Therefore, H3 phosphorylation is involved in processes requiring opposing chromatin states. The level of H3 phosphorylation is mediated by opposing actions of specific kinases and phosphatases during mitosis and gene transcription. The cellular contexts under which specific residues on H3 are phosphorylated in mitosis and interphase are known to some extent. However, the functional consequences of H3 phosphorylation are still unclear.

Biotin is not a natural histone modification.

In addition to its role as the cofactor of biotin-dependent carboxylases, biotin has been demonstrated to have a role in cellular processes including transcription and gene silencing. Histones have been proposed to be modified by biotin in a site-specific manner, providing a pathway by which biotin acts as a regulatory molecule for gene expression. However, there is uncertainty whether biotin attachment to histones in vitro can be extrapolated to biotin as a native histone modification. We critically examined a number of methods used to detect biotin attachment on histones, including [(3)H]-biotin uptake, Western blot analysis of histones, and mass spectrometry of affinity purified histone fragments with the objective of determining if the in vivo occurrence of histone biotinylation could be conclusively established. We found for each of these methods that, while biotin could be readily detected on native carboxylases or histones biotinylated in vitro, biotin attachment on native histones could not be detected in cell cultures from various sources. We conclude that biotin is absent in native histones to a sensitivity of at least one part per 100,000, suggesting that the regulatory impact of biotin on gene expression must be through alternate mechanisms.

Cytogenetic effects induced by Prestige oil on human populations: the role of polymorphisms in genes involved in metabolism and DNA repair.

The spill from the oil tanker Prestige (NW Spain, November 2002) was perhaps the biggest ecological disaster that happened worldwide in the last decades. As a consequence of this catastrophe a general concern led to a huge mobilization of human and technical resources. Given that no information was reported in the scientific literature regarding to the chronic repercussions to human health of exposure to oil spills, a pilot study was performed by our group revealing some increased genotoxic effects in the subjects exposed to the oil during cleaning activities. Due to the seriousness of the results, we extended our study comprising a larger population and including an extensive evaluation of the main polymorphic sites in metabolizing and DNA-repair genes. General increases in micronucleus (MN) frequency and decreases in the proliferation index were observed in individuals with longer time of exposure. Age was a significant predictor of MN frequency. CYP1A1 3'-UTR, EPHX1 codons 113 and 139, GSTP1, GSTM1 and GSTT1 metabolic polymorphisms, and XRCC3 codon 241 and XPD codon 751 repair polymorphisms influenced cytogenetic damage levels. In view of these results, it seems essential to pay more attention to the chronic human health effects of exposure to oil and to focus new studies on such a relevant but overlooked public health field that involves a large number of people all over the world.

Relationship between blood concentrations of heavy metals and cytogenetic and endocrine parameters among subjects involved in cleaning coastal areas affected by the 'Prestige' tanker oil spill.

The sinking of the 'Prestige' oil tanker in front of the Galician coast (NW of Spain) in November 2002 offered a unique opportunity to analyze intermediate cytogenetic and endocrine effects among people exposed to the complex mixture of substances that oil constitutes, including several toxic heavy metals. In this work we evaluated the relationship between exposure to heavy metals (blood concentrations of aluminium, cadmium, nickel, lead and zinc) and genotoxic parameters (sister chromatid exchanges, micronucleus test and comet assay) or endocrine parameters (plasmatic concentrations of prolactin and cortisol) in subjects exposed to 'Prestige' oil during cleaning tasks developed after the spillage. Concentrations of lead were significantly related to the comet assay even after adjusting by age, sex and smoking. Cortisol concentrations were significantly influenced by aluminium, nickel (both, inversely) and cadmium (positively). Women had clearly higher concentrations of prolactin and cortisol, even when adjusting by age, smoking, cadmium, aluminium or nickel. Plasmatic cortisol was jointly influenced by gender, smoking and aluminium or nickel (all p<0.05). In women there was a strong relationship between concentrations of cadmium and prolactin (beta=0.37, p=0.031). When the effects of cadmium, aluminium and nickel on cortisol were simultaneously assessed, only the latter two metals remained statistically significant. Among parameters analysed, cortisol appeared to be the most sensitive to the effects of metal exposure. Plasma levels of cortisol deserve further evaluation as a potentially relevant biomarker to assess the effects of exposure to heavy metals.

Biomonitoring of human exposure to prestige oil: effects on DNA and endocrine parameters.

Since 1960, about 400 tankers spilled more than 377765 tons of oil, with the Prestige accident (Galician coast, NW Spain, November 2002) the most recent. Taking into account the consistent large number of individuals exposed to oil that exists all over the world, it seems surprising the absence in the literature of studies focused on the chronic effects of this exposure on human health. In this work we evaluated the level of DNA damage by means of comet assay, and the potential endocrine alterations (prolactin and cortisol) caused by Prestige oil exposure in a population of 180 individuals, classified in 3 groups according to the tasks performed, and 60 controls. Heavy metals in blood were determined as exposure biomarkers, obtaining significant increases of aluminum, nickel and lead in the exposed groups as compared to controls. Higher levels of genetic damage and endocrine alterations were also observed in the exposed population. DNA damage levels were influenced by age, sex, and the use of protective clothes, and prolactin concentrations by the last two factors. Surprisingly, the use of mask did not seem to protect individuals from genetic or endocrine alterations. Moreover, polymorphisms in genes encoding for the main enzymes involved in the metabolism of oil components were analyzed as susceptibility biomarkers. CYP1A1-3'UTR and EPHX1 codons 113 and 139 variant alleles were related to higher damage levels, while lower DNA damage was observed in GSTM1 and GSTT1 null individuals.

Initial study on the effects of Prestige oil on human health.

The big oil tanker Prestige wrecked at 130 miles from the coast of Galicia, on the Northwest of Spain, in November 19, 2002. During the accident over 40,000 tons of oil were spilled, and along the next weeks 22,000 more reached the shore in the way of three black tides. A great number of people participated in the cleaning tasks. The objective of this study was to initially evaluate the damage caused by Prestige oil in exposed individuals both from the cytogenetic and the endocrine points of view. Exposure level was determined by analysing volatile organic compounds in the environment and heavy metals in blood. Cytogenetic damage was determined by sister chromatid exchanges (SCE), and plasmatic prolactin and cortisol levels were used as biomarkers of endocrine toxicity. Finally we have determined the possible influence of GST genetic polymorphisms (GSTM1 and GSTT1 deletion polymorphisms, GSTP1 Ala105Val) on the evaluated effects. The exposed population was classified according to the performed cleaning tasks in three groups: volunteers that collaborated for 1 week (N=25), hired manual workers (N=20) and hired high-pressure cleaner workers (N=23). The control population consisted of 42 individuals. Exposure to Prestige oil caused cytogenetic damage in exposed individuals, being its effect influenced by age, sex, tobacco consumption and GSTM1 polymorphism. With regard to endocrine toxicity, our results showed that xenobiotics present in Prestige oil induced alterations in hormonal status, and thus it may be considered as an endocrine disruptor. Therefore, the selected parameters have shown to be good indicators of toxicity related to exposure to Prestige oil. In addition, data obtained point to the importance of using protective devices in preventing the effects related to the exposure.

Genetic damage induced by accidental environmental pollutants.

Petroleum is one of the main energy sources worldwide. Its transport is performed by big tankers following some established marine routes. In the last 50 years a total amount of 37 oil tankers have given rise to great spills in different parts of the world, Prestige being the last one. After the accident, a big human mobilisation took place in order to clean beaches, rocks and fauna, trying to reduce the environmental consequences of this serious catastrophe. These people were exposed to the complex mixture of compounds contained in the oil. This study aimed at determine the level of environmental exposure to volatile organic compounds (VOC), and the possible damage induced on the population involved in the different cleaning tasks by applying the genotoxicity tests sister chromatid exchanges (SCE), micronucleus (MN) test, and comet assay. Four groups of individuals were included: volunteers (V), hired manual workers (MW), hired high-pressure cleaner workers (HPW) and controls. The higher VOC levels were associated with V environment, followed by MW and lastly by HPW, probably due to the use of high-pressure cleaners. Oil exposure during the cleaning tasks has caused an increase in the genotoxic damage in individuals, the comet assay being the most sensitive biomarker to detect it. Sex, age and tobacco consumption have shown to influence the level of genetic damage, while the effect of using protective devices was less noticeable than expected, perhaps because the kind used was not the most adequate.

Genotoxicity associated to exposure to Prestige oil during autopsies and cleaning of oil-contaminated birds.

After the accident involving the oil tanker Prestige in November 2002 near 63,000 tons of heavy oil reached Galician coast (Northwest of Spain). This unleashed a large movement of volunteers to collaborate in several cleaning tasks. The aim of this study was to determine whether handling of Prestige oil-contaminated birds during autopsies and cleaning may have resulted in genotoxic damage. We have also evaluated the possible influence of DNA repair genetic polymorphisms (XRCC1 codons 194 and 399, XRCC3 codon 241 and APE1 codon 148) on susceptibility to the genotoxic effects evaluated. Exposure levels were analysed by determining volatile organic compounds in air samples. Peripheral blood samples were obtained from 34 exposed and 35 controls, and comet assay and micronucleus (MN) test were carried out. Genotyping was performed following PCR-RFLP procedures. Results obtained have shown significantly higher DNA damage, but not cytogenetic damage, in exposed individuals than in controls, related to time of exposure. Among exposed individuals, carriers of the variant alleles XRCC1 399Gln and APE1 148Glu have shown altered DNA damage with regard to wild-type homozygotes, suggesting exposure-genotype interactions. No effect of the DNA repair genetic polymorphisms analysed was observed in the MN test.

Assessment of occupational genotoxic risk in the production of rubber tyres.

A broad spectrum of substances is used in the rubber industry, many of them being genotoxic and/or carcinogenic. Convincing evidence of an excess of certain forms of cancer among rubber workers has been provided. The objective of this study was to determine the genotoxic effects in a group of individuals engaged in the production of rubber tyres from a Portuguese factory. Peripheral blood samples were collected from 32 exposed workers and 32 controls, and micronucleus (MN) test, sister chromatid exchanges (SCE) and comet assay were performed. Urinary thioethers were measured as a general biomarker of exposure to electrophilic compounds, and genetic polymorphisms in metabolizing enzymes (CYP2E1 Dra I, EPHX1 codons 113 and 139, GSTP1 codon 105, and GSTM1 and GSTT1 deletion polymorphisms) were analysed as susceptibility biomarkers. Excretion of thioethers was found significantly higher in rubber workers. Also, a non-significant increase in MN frequency related to time of exposure and no effect in SCE were observed in the exposed. Comet assay data showed decreased TL values in the exposed population with respect to the control group, this might indicate the induction of crosslinks by the substances present in the workplace environment. Significant increase in MN frequency was obtained for GSTT1 null exposed individuals with respect to positive ones, and interaction with GSTP1 polymorphism was found. Higher levels of cytogenetic test frequencies were observed in epoxide hydrolase expected low activity donors with respect to medium and high activity individuals. No effect of CYP2E1 or GSTM1 variants was obtained in the biomarkers analysed.

Evaluation of PAH bioaccumulation and DNA damage in mussels (Mytilus galloprovincialis) exposed to spilled Prestige crude oil.

We analyzed the hydrocarbon composition of the Prestige oil as it reached the shores, its solubility in sea water, its bioaccumulation, and the genotoxic damage associated to oil exposure, using Mytilus galloprovincialis as sentinel organism. Mussels were exposed to two oil volumetric ratios (1:500 and 2:500) for 12 days. Great concentrations of total polycyclic aromatic hydrocarbons (TPAH) have been obtained, being in general higher in the samples from the dose of 1:500, both in sea water (55.14 vs. 41.96 microg/l) and mussel tissue (16,993.80 vs. 17,033.00 microg/kg), probably due to the great tendency of these compounds to link to particles in water. Comet assay results reflected an increase in the DNA damage associated to oil exposure, higher in the mussels exposed to the higher aqueous TPAH content. In the view of our results, the importance of the evaluation of biodisponibility, bioaccumulation and DNA damage in the assessment of the effects of xenobiotic pollutants to marine environments could be highlighted.

Influencia de determinados polimorfismos de enzimas metabólicas en la genotoxicidad del estireno / Influences of certain polymorphisms of metabolic enzymes in the genotoxicity of the styrene

El estireno es un compuesto orgánico de gran interés comercial que se utiliza ampliamente en la manufactura de numerosos productos. La exposición a estireno se ha asociado con efectos genotóxicos, fundamentalmente tras activación metabólica a estireno-7,8-óxido (SO). El SO es detoxificado por la epóxido hidrolasa o, en menor grado, por las glutation S-transferasas. En el presente estudio se ha evaluado la influencia de los siguientes factores en la genotoxicidad del estireno: factores fisiológicos y de hábitos de vida, y polimorfismos genéticos en las mencionadas enzimas metabólicas (EPHX1 exones 3 y 4, GSTP1 exones 5 y 6, y los polimorfismos de deleción de GSTM1 y GSTT1). El diseño experimental consistió en exposición de leucocitos periféricos procedentes de 30 donantes sanos a dos dosis de SO, o a un solvente control, y evaluación de la genotoxicidad por medio del test de micronúcleos (MN) y el ensayo del cometa. Los resultados obtenidos muestran que la frecuencia de MN y el daño en el ADN inducidos por el SO se ven influenciados por la edad; sin embargo no se ha detectado influencia del consumo de tabaco, resultando poco claro el efecto del sexo. Se ha observado incremento en la longitud de la cola del cometa a medida que desciende la actividad epóxido hidrolasa en células expuestas a SO, e incremento en la frecuencia de MN en donantes de baja actividad. Estos resultados son consecuentes con la actividad detoxificadora de esta enzima. Además, se han detectado incrementos en la frecuencia de MN para los genotipos GSTP1 *A/*B y *A/*C con respecto a los homocigotos salvajes *A/*A. Esto puede deberse una baja actividad detoxificadora como consecuencia de la afinidad alterada de la proteína variante por el SO. Para los genotipos GSTM1 y GSTT1 no se obtuvieron resultados claros, incluso tras agrupar a los individuos con la misma actividad epóxido hidrolasa esperada, problablemente debido a que la conjugación con glutation juega un papel minoritario en el metabolismo del estireno. (AU)

Effect of epoxide hydrolase and glutathione S-tranferase genotypes on the induction of micronuclei and DNA damage by styrene-7,8-oxide in vitro.

Styrene is one of the most important organic chemicals used worldwide. Its main metabolite, styrene-7,8-oxide (SO), is considered responsible for the genotoxic effects associated with exposure to styrene. SO is detoxified by hydrolysis catalyzed by epoxide hydrolase (EH), or, to a minor extent, by conjugation mediated by glutathione S-transferases (GSTs). The purpose of the present study was to investigate whether EH (exons 3 and 4), GSTP1 (exons 5 and 6), GSTM1 and GSTT1 polymorphisms have any influence on the genotoxicity of SO in human leukocytes. Peripheral leukocytes from 30 healthy donors were exposed to SO (50 and 200 micro M) and genotoxicity was evaluated by means of the micronucleus (MN) test and alkaline comet assay, using 1% DMSO as solvent control. When EH genotypes were classified in low, medium, and high with respect to the expected EH activity, an increase in induced comet tail length was observed with decreasing EH activity in SO-exposed cells. An increase was seen in induced MN frequency in EH low-activity donors. These findings are consistent with the detoxifying activity of this enzyme. In addition, increases in MN frequencies for GSTP1 *A/*B and *A/*C genotypes with regard to the wild-type homozygous *A/*A genotype were detected. This may be due to a low detoxifying activity as a consequence of altered SO affinity of the variant protein, but must be confirmed using homozygote variant individuals, not included in this study. No clear results were obtained for GSTM1 or GSTT1 genotypes, even when performing the analysis after grouping individuals with the same expected EH activity, probably due to the minor role that glutathione conjugation plays in styrene metabolism. The present in vitro findings using human leukocytes suggest that polymorphisms in EH, and, to a lesser extent, in GSTP1, may influence induction of cytogenetic and DNA damage by SO.

Individual sensitivity to DNA damage induced by styrene in vitro: influence of cytochrome p450, epoxide hydrolase and glutathione S-transferase genotypes.

Styrene is a monomer of great commercial interest; its polymers and copolymers are used in a wide range of applications. In humans, styrene metabolism involves oxidation by cytochrome p450 monooxygenases (CYPs) to styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure and detoxification by means of epoxide hydrolase (EH) and glutathione S-transferases (GSTs). The objective of this study was to investigate if genetic polymorphisms of metabolic enzymes modulate styrene-induced DNA damage in human leukocytes. CYP2E1, CYP1A1, EH, GSTP1, GSTM1 and GSTT1 polymorphisms were determined in 30 healthy donors and alkaline comet assay was carried out in isolated leukocytes exposed to 5 and 10 mM styrene, using 1% acetone as solvent control. The results obtained suggest that CYP1A1 m1, m2 and m4, CYP2E1 Dra I and GSTP1 (exons 5 and 6) polymorphisms may affect styrene induction of DNA damage in human leukocytes.