Morbility, clinical data and proteomic analysis of IUGR and AGA newborns at different gestational ages.

The data are related to the proteomic analysis of 43 newborns with intrauterine growth retardation (IUGR) and 45 newborns with appropriate weight for gestational age (AGA) carried out by separation via 2DE and analyzed by MS-TOF/TOF. All newborns were separated into three gestational age groups, "Very Preterm" 29-32 weeks, "Moderate Preterm" 33-36 weeks, and, "Term" ≥37weeks. From each newborn, blood was drawn three times from birth to 1 month life. High-abundant serum proteins were depleted, and the minority ones were separated by 2DE and analyzed for significant expression differences. The data reflect analytic and clinic variables analyzed globally and categorized by gestational age in relation to IUGR and the optimization of conditions for 2-DE separation. The data from this study are related to the research article entitled "Alterations of Protein Expression in Serum of Infants with Intrauterine Growth Restriction and Different Gestational Ages" (M.D. Ruis-González, M.D. Cañete, J.L. Gómez-Chaparro, N. Abril, R. Cañete, J. López-Barea, 2015) [1]. The present dataset of serum IUGR newborn proteome can be used as a reference for any study involving intrauterine growth restriction during the first month of life.

Combination of direct infusion mass spectrometry and gas chromatography mass spectrometry for toxicometabolomic study of red blood cells and serum of mice Mus musculus after mercury exposure.

Although mercury (Hg) is an important environmental and occupational pollutant, its toxicological effects, especially in serum and red blood cells (RBCs), have been scarcely studied. A toxicometabolomics workflow based on high resolution mass spectrometry approaches has been applied to investigate the toxicological effects of Hg in Mus musculus mice after subcutaneous injection for 10 days, which produced inflammation and vacuolization, steatosis and karyolysis in the hepatic tissue. To this end, direct infusion mass spectrometry (DIMS) of polar and lipophilic extracts from serum and RBCs, using positive and negative mode of acquisition (ESI+/ESI-), and gas chromatography-mass spectrometry were used. A quantitative analysis of reversible oxidized thiols in serum proteins demonstrated a strong oxidative stress induction in the liver of Hg-exposed mice. Endogenous metabolites alterations were identified by partial least squares-discriminant analysis (PLS-DA). Mercury-exposed mice show perturbations in energy metabolism, amino acid metabolism, membrane phospholipid breakdown and oxidative stress-related metabolites in serum along the exposure. This work reports for the first time the effects of Hg-exposure on RBCs metabolic pathways, and reveals disturbances in glycolysis, membrane turnover, glutathione and ascorbate metabolisms.

Use of metallomics and metabolomics to assess metal pollution in Doñana National Park (SW Spain).

Monitoring organism exposure to heavy metals has acquired increased importance in the last decades. The mouse Mus spretus has been used to assess the biological response to contaminants in the relevant ecological area of Doñana National Park (DNP) and surrounding areas (SW Spain), where many migrating birds land for breeding and feeding every year. A metallomics approach, based on the characterization of metal biomolecules using size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and a metabolomics approach based on direct infusion to a mass spectrometer (DI-ESI-QTOF-MS) followed by a partial linear square-discriminant analysis (PLS-DA), were used to compare the biological responses of M. spretus living in three areas of DNP (the reference) and surrounding areas (El Partido and El Matochal). The activities of key antioxidant enzymes, such as Cu/Zn-SOD, Mn-SOD, CAT, GR, and guaiacol peroxidase, were also determined in connection with environmental contamination issues. The results show differences caused by the presence of metals in the ecosystem that affected to the levels of metals and metalloproteins, such as MT, Cu/Zn-SOD, or Mn-CA, the breakdown of membrane phospholipids, perturbations in metabolic pathways, related to energy metabolism, and oxidative stress.

Evolution of metallotionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposure.

Characterization of Cd-binding proteins has great analytical interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatography: size exclusion followed by reversed-phase high performance liquid chromatography, to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure experiments to Cd. Exposure experiments were carried out by subcutaneous injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body weight per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes-MTs along cadmium exposure.

Biological response of free-living mouse Mus spretus from Doñana National Park under environmental stress based on assessment of metal-binding biomolecules by SEC-ICP-MS.

A metallomic approach based on the use of size-exclusion chromatography (Superdex-75) with inductively coupled plasma mass spectrometry (ICP-MS) detection is combined with anion or cation exchange chromatography to characterize the biological response of the free-living mouse Mus spretus. The approach has been applied to contaminated and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas affected by differential contamination from mining, industrial, and agricultural activities have been considered. The high presence of Mn, Cu, and Zn in liver and As and Cd in kidney is remarkable, especially in contaminated areas. The size exclusion chromatograms traced by Mn in liver cytosolic extracts are more intense than in kidney; a Mn-peak matching with the standard of 32 kDa (superoxide dismutase) is present in these organs, and its intensity is correlated with the concentration of Mn in the extracts. High-intensity peaks traced by Cu, Zn, and Cd at 7 kDa (matching with metallothionein I standard) in liver extract are triggered by the presence of contaminants. Other peaks related with molecules of 32 and 67 kDa traced by Cu and Zn can also be observed, although their intensity is higher in sites with low contamination. In kidney extracts, the presence of a Cd-peak with Mr of 7 kDa (tentatively Cd-metallothionein) with high intensity under the action of contaminants was observed, but high biological responses are also proven in the protected area of the Park, which denotes a progressive increase of diffuse contamination.

Relationship of oxidative stress and endothelial dysfunction in sleep apnoea.

The aim of the present study was to evaluate ischaemic reactive hyperaemia (IRH) in obstructive sleep apnoea (OSA) and its relationship with oxidative stress. We studied 69 consecutive patients referred to our Sleep Unit (Reina Sofia University Hospital, Cordoba, Spain). Patients with chronic diseases or those taking medication were excluded. IRH was assessed before and after polysomnography. Morning IRH and oxidative stress markers were compared between patients with (apnoea-hypopnoea index (AHI) ≥ 5) and without (AHI < 5) OSA. Measurements were repeated in 25 severe OSA patients after continuous positive airway pressure (CPAP) therapy. We included 46 OSA patients (mean ± sd AHI 49 ± 32.1) and 23 non-OSA subjects (AHI 3 ± 0.9). The OSA patients showed a significant worsening of morning IRH, and a significant increase in malondialdehyde and 8-hydroxydeoxyguanosine levels. Only the oxygen desaturation index independently explained morning IRH, while malondialdehyde levels showed a weak effect on IRH. In severe OSA patients, IRH improved significantly after CPAP treatment, as did malondialdehyde, 8-hydroxydeoxyguanosine and protein carbonyl levels. In OSA patients, endothelial dysfunction and oxidative stress were observed, and IRH worsened after sleep. The increase in oxidative stress was not associated with IRH, while intermittent hypoxia was strongly associated with IRH. In severe OSA patients, CPAP treatment improved oxidative stress and endothelial function.

Alterations to proteome and tissue recovery responses in fish liver caused by a short-term combination treatment with cadmium and benzo[a]pyrene.

The livers of soles (Solea senegalensis) injected with subacute doses of cadmium (Cd), benzo[a]pyrene (B[a]P), or their combination, were screened for alterations to cytosolic protein expression patterns, complemented by cytological and histological analyses. Cadmium and B[a]P, but not combined, induced hepatocyte apoptosis and Kupfer cell hyperplasia. Proteomics, however, suggested that apoptosis was triggered through distinct pathways. Cadmium and B[a]P caused upregulation of different anti-oxidative enzymes (peroxiredoxin and glutathione peroxidase, respectively) although co-exposure impaired induction. Similarly, apoptosis was inhibited by co-exposure, to which may have contributed a synergistic upregulation of tissue metalloproteinase inhibitor, beta-actin and a lipid transport protein. The regulation factors of nine out of eleven identified proteins of different types revealed antagonistic or synergistic effects between Cd and B[a]P at the prospected doses after 24 h of exposure. The results indicate that co-exposure to Cd and B[a]P may enhance toxicity by impairing specific responses and not through cumulative damage.

Proteomics of juvenile senegal sole (Solea senegalensis) affected by gas bubble disease in hyperoxygenated ponds.

Solea senegalensis is a commercial flat fish traditionally farmed in earth ponds in coastal wetlands that might also become important to more intensive aquaculture. Gas bubble disease (GBD) is a potential risk for outdoor fish farming, particularly in certain periods of the year, related to improper management leading to macroalgae blooms. Physical-chemical conditions inducing hyperoxia, including radiation, temperature, and high levels of dissolved oxygen, have been monitored in fish affected by GBD together with observed symptoms. Exophthalmia, subcutaneous emphysemas, obstruction of gill lamellae, hemorrhages, and anomalous swimming were the main effects of oxygen supersaturation. A proteomic study was carried out for the first time under aquaculture conditions and protein expression changes are described for fish that were subject to hyperoxic conditions. Proteins identified in gill of GBD-affected fish are related to oxidative alteration of cytoskeleton structure/function (beta-tubulin, beta-actin), motility (light myosin chain, alpha-tropomyosin), or regulatory pathways (calmodulin, Raf kinase inhibitor protein), reflecting the central role of gill in oxygen exchange. Hepatic proteins identified are related to protein oxidative damages (beta-globin, FABPs), protection from oxidative stress (DCXR, GNMT), and inflammatory response (C3), in agreement with the predominant metabolic role of liver. Comparison of protein expression patterns and protein identification are suggested as potentially specific hyperoxia biomarkers that would facilitate prevention of GBD outbreaks.

Metal-binding molecules in the organs of Mus musculus by size-exclusion chromatography coupled with UV spectroscopy and ICP-MS.

Mus musculus mice have been investigated for the total elements content in different organs (lung, liver, spleen, kidney, brain, testicle, heart and muscle) and molecular mass distribution patterns of Mn, Ni, Cu, Zn, As, Pb, Cr, Fe, Co, Se and Cd. Some differences have been found in the organs studied, with especially relevant being the Cu-containing fraction present only in the brain and the As-containing one in the liver. Other differences related to the abundance of the metallospecies have also been found. The present paper is the first step in the study of the "metallome" of this inbred laboratory species from which the genome is completely known. This organism could be used as a model in future studies focused on wild mice and the analytical approach developed could be applied to wild mice to find markers of environmental pollution. [figure: see text] The present paper is the first step in the study of the "metallome" of the inbred laboratory specie Mus musculus from which the genome is completely known. Some interesting differences have been found in the extracts from the organs that are discussed along the text.

Esterases as pesticide biomarkers in crayfish (Procambarus clarkii, Crustacea): tissue distribution, sensitivity to model compounds and recovery from inactivation.

The specific activities of acetyl- and butyrylcholinesterase and carboxylesterase were assayed in the digestive gland and in nervous and muscle tissues of the crayfish Procambarus clarkii. Since acetylcholinesterase prevails in nervous tissue and carboxylesterase in digestive gland, they are proposed as biomarkers. Muscle had negligible activities of all esterases, and all tissues had a low butyrylcholinesterase activity. Esterases were mostly cytosolic in digestive gland and muscle, but membrane-bound in nervous tissue; use of Triton X-100 is not recommended due to its widely diverging effects in esterase assays. Phenylmethylsulphonylfluoride inhibited acetyl- and butyrylcholinesterase in extracts from all tissues, and in digestive gland only carboxylesterase. In digestive gland, tetra[monoisopropyl]-pyrophosphorotetramide inhibited all esterases with different sensitivities, while in muscle and nervous tissue it only partially inhibited all esterases. Carbamates inhibited 100-fold more strongly than organophosphates acetyl- and butyrylcholinesterase activities. Carboxylesterase was inhibited by carbaryl and chlorpyrifos, but not by eserine and malathion. In vitro conditions to evaluate recovery from inactivation of esterases by model pesticides were established for acetylcholinesterase and carboxylesterase. The new reactivation protocol could be useful as a biomarker of pesticide exposure to differentiate between dilution-reversible inhibitions, indicating carbamate exposure, from dilution-irreversible effect, attributed to organophosphate exposure.

Doñana National Park survey using crayfish (Procambarus clarkii) as bioindicator: esterase inhibition and pollutant levels.

Utility of carboxylesterase and acetylcholinesterase inhibition as pesticide exposure biomarker was studied at Doñana National Park (SW Spain) in crayfish (Procambarus clarkii). Activities were measured in animals from reference sites or potentially exposed to pesticides, and their reactivation studied after dilution or 2-PAM treatment. Crayfish from affected sites had significantly less carboxylesterase and acetylcholinesterase activity than reference ones. No significant differences were found after dilution or 2-PAM treatment, showing that inhibition was irreversible. High pesticide levels were found in water and/or soil at rice growing sites, and lower levels at other affected places. High metal levels existed at rice growing sites and lower at other affected and at both reference sites. A combined effect on esterase inhibition of pesticides and metals is proposed. This field study suggest that the rice growing areas near Guadiamar stream are most polluted, followed by strawberry and citrics growing zones near Partido and Rocina streams. However, no correlation exist between the pesticide concentration at different sites and the extent of esterase inhibition, indicating that other factors could affect esterase response of animals from polluted sites.

Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic littoral.

Metal accumulation and some of their biochemical effects have been studied in oysters (Crassostrea angulata) and mussels (Mytilus galloprovincialis) of the South Atlantic Spanish littoral. Especial attention has been paid to antioxidant defences and oxidative damage to biomolecules. Deep differences in the response of oysters and mussels to metal pollution were found. Oysters, with the higher metal loads of both species, showed increased antioxidant defences, and less extensive oxidative damage. In contrast, mussels, which accumulated much lower metal concentrations, showed clear increases in oxidized biomolecules, in agreement with their low increases in the antioxidant defence mechanisms. Our results suggest that mussels are more sensitive and less well adapted to metal pollution, probably explaining their absence in the most contaminated studied site, Mazagón. We conclude that oysters can be used as more sensitive bioindicator of pollution in the South Spanish littoral, and as a suitable model to study the adaptation to metal pollution.

Evolution of biological effects of Aznalcóllar mining spill in the Algerian mouse (Mus spretus) using biochemical biomarkers.

The status of Guadiamar stream, polluted in 1998 by metals spilled from a pyrite tailings dam, was monitored from 1999 to 2001 to assess possible biological effects in terrestrial ecosystems of Doñana National Park (DNP) (Andalusia, SW Spain). The Algerian mouse (Mus spretus) was used as bioindicator at different Guadiamar and Doñana sites. Eleven biochemical parameters, including the activities of antioxidative and biotransforming enzymes and oxidative damages to biomolecules, were assayed in liver as biomarkers responsive to metals and organic pollutants. In 2001, metals were also determined in kidney and their possible correlation with biomarker responses was studied. Contents of Pb, Cd and As significantly correlated with several antioxidative enzymes. Biomarkers responsive to oxidative stress indicate the presence of transition metals in the high and medium Guadiamar course, and their response diminished with the distance to the collapsed dam. The high ethoxyresorufin-O-deethylase (EROD) activity of mice from the medium and low Guadiamar course point to organic pollutants, such as the pesticides used in intensive crops grown in areas nearby Doñana. The increasing responses of several biomarkers at reference sites may suggest a progressive pollution of key Doñana ecosystems.

Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata).

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.

Uptake and clearance of PCB congeners in Chamaelea gallina: response of oxidative stress biomarkers.

PCB uptake and clearance by clams, Chamaelea gallina, were studied in specially designed flow-through channels. After 8 weeks exposure to 10 ppb Aroclor 1254 in water, clams were depurated for 10 weeks, in the same exposure channel or after transfer to clean systems. Accumulation of the 20 congeners studied depended on its initial abundance and physicochemical properties. A linear relationship was found between log bioconcentration factor and log octanol/water partition coefficient of each form. Clearance of each PCB depended also on its initial load and solubility, being faster in clams transferred to clean systems. Exposure significantly enhanced catalase and 6-P-gluconate dehydrogenase activities, but not other antioxidative enzymes. Superoxide dismutase, low during the exposure phase, increased seven-fold during depuration. Aroclor-treated clams had higher GSH levels than controls, but decreased to 15-35% after 2 days clearance, rose to 150% after 12 days, and declined to low levels by the end of the experience. Biotransformation of PCBs to quinones and redox cycling-promoted oxidative stress might explain the increased antioxidative defenses. The biochemical changes observed at the beginning of clearance could be attributed to clam handling, by adaptation to and recovery from hypoxic/anoxic stress.

New methods to use fish cytochrome P4501A to assess marine organic pollutants.

A new methodology has been developed to assess cytochrome P4501A expression in two South Atlantic Spanish fish, guilthead seabream (Sparus aurata) and grey mullet (Liza aurata), used as pollution bioindicators. Degenerate oligos were used to amplify by reverse transcription and PCR (RT-PCR) specific cyp1A cDNA sequences, used subsequently to design specific primers to get the full cDNA by rapid amplification of cDNA ends. A new assay has been developed to quantitate cyp1A expression by RT-PCR in an automated DNA sequencer. The effect of beta-naphthoflavone inducing biotransformation has been used to compare three distinct pollution biomarkers: EROD activity, ELISA determination of CYP1A, and 2-aminoanthracene (2-AA) activation. Immunodetection by ELISA or Western blot was inconsistent in S. aurata and L. aurata. EROD activity yielded satisfactory results; the higher induction was observed by bioactivation of 2-AA to mutagens detected with strain BA149 of Salmonella typhimurium, in agreement with the high sensitivity previously described for this biomarker. The present paper summarizes the current status of our research.

Content of 8-oxodG in chromosomal DNA of Sparus aurata fish as biomarker of oxidative stress and environmental pollution.

The 8-oxodG content has been measured in chromosomal DNA of gilthead seabream (Sparus aurata) by HPLC-EC. Susceptibility of different tissues to oxidative DNA damage was studied by exposing fish to model pollutants. Cu(II), paraquat (PQ) and malathion failed to promote DNA oxidation in liver, while dieldrin significantly increased the 8-oxodG content in this organ, but not in gills or blood. After PQ exposure, fish liver showed high levels of glucose-6-P dehydrogenase (G-6PDH) and GSSG reductase activities. The increased antioxidant status and the lack of a specific transport system could explain the lack of susceptibility of liver to DNA oxidative damage induced by PQ. Increased levels of 8-oxodG were detected in the gills of PQ-exposed fish after 8 and 24 h. In contrast, after 48 h exposed fish contained lower 8-oxodG levels than controls. The existence of a PQ transport system in this O2-rich organ and the lack of a significant increase in antioxidant defenses would explain the sensitivity of gills to DNA damage promoted by PQ. Elimination of this soluble chemical and the putative induction of DNA-repair enzymes specific for oxidative damages could explain the drop of 8-oxodG levels at longer times. Fish exposed to moderate levels of urban and industrial pollution showed significantly high 8-oxodG content in hepatic DNA. We conclude that 8-oxodG determination in chromosomal DNA by HPLC-EC is a potentially useful biomarker of environmental pollution, although its response is still somewhat lower than that of other well-established biomarkers of oxidative stress.

Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences.

This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences. The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H2O2 or O2.- or the DNA repair protein for 8-oxoG. Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H2O2 in K medium (1% casamino acids and 1% glucose), as compared with nutrient broth. This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities. Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions. Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration. Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions. Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H2O2 exposure. Catalase deficiency resulted in a dramatic decrease in viability after H2O2 exposure. A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality, though to lesser extent than a deficiency of catalase activity. However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H2O2 treatment.

In vivo transcription of nrdAB operon and of grxA and fpg genes is triggered in Escherichia coli lacking both thioredoxin and glutaredoxin 1 or thioredoxin and glutathione, respectively.

We have previously described () that Escherichia coli maintains a balanced supply of deoxyribonucleotides by a regulatory mechanism that up-regulates the levels of ribonucleotide reductase with the lack of its main hydrogen donors thioredoxin, glutaredoxin 1, and glutathione (GSH). By using a semi-quantitative reverse transcription/multiplex polymerase chain reaction fluorescent procedure that enables simultaneous analysis of up to seven mRNA species, we now demonstrate that regulation operates at the transcriptional level. Double mutant cells lacking both thioredoxin and glutaredoxin 1 had increased transcription of the nrdAB operon, as compared with the corresponding wild type parent (maximal induction of 10- and 9-fold for mRNA of nrdA and nrdB genes, respectively). Likewise, a dramatic increase of 36-fold in grxA mRNA was observed in bacteria simultaneously deficient in thioredoxin and GSH (the physiological reductant of all glutaredoxins). The increased expression of the grxA gene in trxA gshA double mutant bacteria was mimicked in trxA single mutant cells by depletion of GSH with diethylmaleate (DEM). This induction of grxA transcription was rapid since maximal increase was detected upon 10 min of DEM exposure. Like grxA expression, the basal level of fpg mRNA, encoding formamidopyrimidine-DNA glycosylase, was increased (about 4-fold) in a trxA gshA double mutant strain; this expression was also induced upon exposure to DEM (11-fold maximal induction). These results suggest that transcription of grxA might share common redox regulatory mechanism(s) with that of the fpg gene, involved in the repair of 8-oxoguanine in DNA.

Mutagen content and metabolic activation of promutagens by molluscs as biomarkers of marine pollution.

Organisms combat pollutants by inducing biotransformation pathways, which can be used for biomonitoring. Several parameters--biomarkers--change in stressed organisms or populations at different organisation levels. Molecular or cellular biomarkers are early-warning indicators of pollution. Xenobiotics are first biotransformed by phase I enzymes and then conjugated with endogenous metabolites by phase II enzymes. Many organic xenobiotics are initially biotransformed by cytochrome P4501A1; in mammals, it is induced by pollutants via Ah receptor, although in marine invertebrates, its inducibility is much more equivocal. Metallothioneins are small Cys-rich proteins which bind transition metals; they detoxicate pollutant metals and are clearly induced in metal-exposed marine invertebrates. Some pollutants are genotoxins or can be converted into them. Determination of mutagens in bivalve molluscs following extraction with solvents and assay of mutagenicity with bacterial tests is a useful biomarker for marine pollution. While some pollutants are directly mutagenic, others are only mutagenic after they are activated to mutagenic derivatives by monooxygenases or conjugative enzymes. Many of these catalysts are induced by xenobiotics; thus, increased activation of known promutagens can be used as biomarker of environmental pollution. Bioactivation of promutagens requires the simultaneous action of different pathways, thus, reproducing more closely the in vivo situation than the specific assay of individual biotransforming enzymes. Study of molluscs with different pollution levels indicates that polluted animals have higher capacity to activate 2-aminoanthracene and contain more apolar mutagens than those from reference areas.