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.

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.

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.

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.

Purification and properties of bovine thioredoxin system.

Using a variety of chromatographic techniques, a crude extract from bovine liver was fractionated to obtain pure preparations of thioredoxin reductase, thioredoxin, glutaredoxin and glutathione reductase with good yields. The turbidimetric assay of thioredoxin with insulin as the disulfide substrate was optimized; by incorporation of the lag time (tau) into the calculations, linearity was maintained for a wider range of thioredoxin concentrations, and a distinction could be made between reduced and non-reduced forms. Subunit composition and molecular mass, absorption spectrum and kinetic parameters of thioredoxin reductase were similar to those of other mammalian thioredoxin reductases. By chromatofocusing, two peaks of activity were detected at pH 5.5 and 5.8. Structural changes undergone by the thioredoxin molecule upon oxido-reduction were detected by isoelectric focusing, with a shift of 0.1 pH unit of its pI, and by analytical anion exchange chromatography, with a conspicuous shift of its retention time. These two methods also revealed the presence of a form of thioredoxin not undergoing the above mentioned redox-mediated structural shifts that accounted for > 75% of the total activity.

Immunolocalization of thioredoxin and glutaredoxin in mammalian hypophysis.

Thioredoxin (TRX) and glutaredoxin (GRX) are two small proteins catalyzing thiol-disulfide oxidoreductions. A role of both proteins in secretory processes has been suggested and recently it has been demonstrated that thioredoxin functions as a growth factor for lymphocytes in cell cultures. Here we report on the immunolocalization by light microscopy of both proteins in the hypophysis of mammals. We have used affinity purified specific antibodies that give a single band on immunoblots against crude extracts from pig and calf neurohypophysis and adenohypophysis. Thioredoxin was prominently localized in the folliculo-stellatae cells of the adenohypophysis while only a minor proportion of the glandular cells were positive. In the neurohypophysis, thioredoxin immunoreactivity was very intense in the pituicytes and moderate in the clusters of synaptic terminals. Glutaredoxin localization in the adenohypophysis resembled that of thioredoxin whereas in the neurohypophysis there was a clear differential localization: the neurosecretory terminals and Herring bodies were intensely stained for glutaredoxin but not the pituicytes. These results suggest that thioredoxin may be involved in the paracrine modulatory action of folliculo-stellatae cells and that these cells and pituicytes may have similar functions in their respective parts of the hypophysis; the association of glutaredoxin with secretory processes is further documented.

Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants.

Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism. To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used. Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats. The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system. This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics. The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic. The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity. The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.

Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts.

Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5-20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or "Nature's pesticides" (the major toxic chemicals ingested by phytoplankton filter-feeders), and/or the result of human activities remains to be determined.

Promutagen activation by fish liver as a biomarker of littoral pollution.

Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1-, 18.6- and 42.8-fold higher capability to activate benzo(a)pyrene, 2-acetyl-aminofluorene and 2-aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose-response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2-aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments.

Mutagenic activation of aromatic amines by molluscs as a biomarker of marine pollution.

Mutagenic activation of arylamines by mollusc S9 fractions was evaluated as a biomarker for marine pollution. Two bivalve species were used as bioindicators, the common mussel (Mytilus edulis) and the striped venus (Chameleo gallina). A strain of Salmonella typhimurium overproducing O-acetyltransferase was used as indicator of mutagenicity. Mussels from an area of the North Atlantic Spanish zone that was exposed to an accidental crude oil spill were compared to bivalves from a reference area. C. gallina samples were from low polluted and highly polluted areas of the South Atlantic Spanish littoral. The promutagen 2-aminoanthracene (2-AA) was activated to mutagenic derivative(s) by S9 fractions from both C. gallina and M. edulis. Animals from contaminated sites showed higher arylamine activation capabilities than reference animals. This was further correlated with the mutagenic activities of corresponding cyclopentone-dichloromethane animal extracts. 2-AA activation by mollusc S9 was potentiated by alpha-naphthoflavone (ANF), known to inhibit PAH-inducible CYP1A cytochromes from vertebrates, but inhibited by methimazole (MZ), a substrate of the flavin monooxygenase (FMO) system. 2-AA-activating enzymes were mainly cytosolic; this localization clearly suggests that such activity could be attributed to soluble enzymes, different from the CYP1A or FMO systems. In conclusion, mutagenic activation of arylamines by mollusc S9, using as indicator a strain of Salmonella typhimurium that overproduces O-acetyltransferase, could be a reliable biomarker for marine 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.

Oxidative stress in fish exposed to model xenobiotics. Oxidatively modified forms of Cu,Zn-superoxide dismutase as potential biomarkers.

Fish (Sparus aurata) were intraperitoneally injected with model xenobiotics and several biomarkers of oxidative stress were analysed after 2 and 7 days exposure. The levels of soluble thiobarbituric acid reactive substances (TBARS) increased markedly in animals treated with polar xenobiotics, CuCl2 or paraquat; exposure to the apolar xenobiotics, dieldrin or malathion, enhanced significantly the microsomal TBARS while decreasing the microsomal glutathione transferase activity. The specific superoxide dismutase (SOD) activity increased in Cu(II)-injected animals but diminished in fish exposed to paraquat. After isoelectrofocusing separation and activity staining cell-free extracts of fish exposed to Cu(II), dieldrin or malathion displayed two new Cu,Zn-SOD isoforms of intermediate pI. An additional Mn-SOD was observed in dieldrin-injected fish, but only a faint new acidic isoform was observed in paraquat-injected animals. The new SOD bands were reproduced in vitro by incubation of cell-free extracts with systems generating superoxide anion or hydrogen peroxide and with a tert-butyl hydroperoxide/ADP-Fe system. Metallothionein induction was observed in Cu(II) or paraquat-exposed fish, but not in animals injected with apolar xenobiotics. So, the new SOD bands are possibly oxidized forms of this enzyme and can be considered as useful early biomarkers of oxidative stress due to transition metals or organic xenobiotics.

Incubation of superoxide dismutase with malondialdehyde and 4-hydroxy-2-nonenal forms new active isoforms and adducts. An evaluation of xenobiotics in fish.

The effects in fish (Sparus aurata) of dieldrin, previously reported to be an inducer of peroxisomal enzymes (Pedrajas et al., Comp. Biochem. Physiol. 115C (1996) 125-131), were compared with those of clofibrate. Although dieldrin provoked the more severe peroxisomal changes, both compounds induced oxidative stress as detected by the increased levels of microsomal thiobarbituric acid reactive substances; however the malondialdehyde (MDA) content, determined after HPLC separation of the MDA-TBA complex, was not significantly altered. These results suggest that, besides MDA, other aldehydes were formed in xenobiotic-injected fish, leading us to assess the oxidative effects of such xenobiotics by following changes in superoxide dismutase (SOD) pattern. New active SOD isoforms were detected by isoelectrofocusing in the light mitochondrial (LMF) and cytosolic (CF) fractions. Most of the new SOD bands could be reproduced in vitro by incubation of fish liver cell-free extracts with MDA. To clarify the effects of aldehydes, Cu,Zn- and Mn-SOD isoforms were purified and amino acid analysis was carried out. The new bands found in LMF and CF fractions were reproduced in vitro after incubation of pure SODs with MDA and 4-hydroxy-2-nonenal (HNE), the new SOD bands formed being coincident with the loss of Lys or His residues. Lysine residues were preferentially derivatized after treatment of Cu,Zn-SOD with MDA, but in Mn-SOD the lysine residues were modified only after treatment with MDA, while the histidine residues were modified only by HNE. No change of SOD activity was detected after MDA or HNE exposure, although at the higher aldehyde concentrations used protein aggregates were formed. Therefore, the appearance of new active SOD bands, after isoelectrofocusing separation, can be proposed as a biomarker of oxidative stress.

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.

The L-arabinose-resistance test with Salmonella typhimurium strain SV3 selects forward mutations at several ara genes.

A new assay has been described for mutagenicity testing using an L-arabinose-sensitive strain of Salmonella typhimurium. The test strain SV3 and several L-arabinose-resistant mutants selected therefrom are characterized in the present study by 3 different criteria: inhibition of growth by L-arabinose, accumulation of keto-sugars, and activities of the enzymes involved in L-arabinose catabolism. Strain SV3 (ara-531) shows high levels of inducible L-arabinose isomerase (EC 5.3.1.4) and L-ribulokinase (EC 2.7.1.16) activities, but is deficient in L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4), the enzyme encoded in Escherichia coli by gene D in the araBAD operon. Addition of L-arabinose to SV3 growing in glycerol or casamino acids stops growth. D-Glucose only partially reverses this inhibition. Reversion of the ara-531 mutation restores different levels of epimerase activity and resistance to L-arabinose. However, the great majority of the L-arabinose-resistant mutants do not utilize L-arabinose. The physiological and enzymatic properties of these L-arabinose non-utilizing mutants suggest that L-arabinose resistance is due to forward mutations in at least 3 other genes, araA, araB and araC, blocking steps prior to L-ribulose 5-phosphate accumulation.