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.

Hypersensitivity and desensitization to antineoplastic agents: outcomes of 189 procedures with a new short protocol and novel diagnostic tools assessment.

BACKGROUND: Desensitization to antineoplastic agents is becoming a standard of care. Efforts to establish and improve these techniques are being made at many institutions. Our aims are to evaluate a new rapid desensitization protocol designed to be shorter (approximately 4 h) and safer (reducing hazardous drugs exposure risks) and to assess the oxaliplatin-specific immunoglobulin E (IgE) as a novel diagnostic tool. METHODS: Prospective, observational, longitudinal study with patients who, for a 1-year period, suffered reactions to antineoplastic agents and were referred to the Desensitization Program at Ramon y Cajal University Hospital (RCUH). Patients were included or excluded as desensitization candidates after anamnesis, skin testing, risk assessment, and graded challenge. Specific IgE was determined in oxaliplatin-reactive patients. Candidate patients were desensitized using the new RCUH rapid desensitization protocol. RESULTS: Of 189 intravenous rapid desensitizations, 188 were successfully accomplished in the 23 patients who met inclusion criteria for desensitization (of 58 referred patients). No breakthrough reactions occurred in 94% of desensitizations, and most breakthrough reactions were mild. In 10 oxaliplatin-reactive patients, 38 desensitizations were successfully accomplished. Sensitivity for oxaliplatin-specific IgE was 38% (0.35UI/l cutoff point) and 54% (0.10UI/l cutoff point); specificity was 100% for both cutoff points. CONCLUSIONS: In the hands of a Desensitization Program, managed by drug desensitization experts, this new protocol has proven an effective therapeutic tool for hypersensitivity to several antineoplastic agents (oxaliplatin, carboplatin, paclitaxel, docetaxel, cyclophosphamide, and rituximab); moreover, it improves safety handling of hazardous drugs. We report the first large series of oxaliplatin desensitizations. Oxaliplatin-specific IgE determination could be helpful.

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.

A descriptive study of transthyretin amyloidosis in a tertiary hospital without a referral unit.

BACKGROUND AND OBJECTIVE: Transthyretin amyloidosis (ATTR) is a rare disease that is part of systemic amyloidosis and is life-threatening. It can affect all organs and systems, the most frequent being neurological and cardiac involvement. This study aims to detect possible ATTR cases and carry out a descriptive study of them. MATERIAL AND METHODS: Descriptive single-centre study carried out in a tertiary hospital, which included patients with suspected ATTR between September 2016 and January 2020. RESULTS: A total of 190 suspected ATTR patients were detected. The study includes 100 of these patients, as well as 10 relatives of patients in whom ATTR was detected in its genetic variant (ATTRv). In total, ATTRv was detected in 7 individuals (3 with a presymptomatic mutation of the disease), 16 patients with age-related ATTR and 31 individuals with unknown cardiac amyloidosis with the tests performed, which confirms the presence of this disease in non-endemic areas. CONCLUSIONS: ATTR is a disease that must be taken into account in the differential diagnosis of patients with heart failure with preserved LVEF, especially if associated with neurological symptoms.

Atypical presentation of transthyretin amyloidosis in a non-endemic area. / Presentación atípica de amiloidosis por transtiretina en un área no endémica.

BACKGROUND AND OBJECTIVE: There are 2 types of amyloidosis caused by transthyretin deposits: the wild type (wt-ATTR) and the mutant type (m-ATTR), transmitted by autosomal dominant inheritance with variable penetrance, manifesting with neurological and/or cardiac symptoms. We report on 3 families affected by m-ATTR diagnosed in a nonendemic area. MATERIAL AND METHODS: We studied 63 patients with a high suspicion of ATTR. The diagnosis was subsequently performed by magnification through polymerase chain reaction of DNA. For the positive cases, we studied the first-degree relatives. RESULTS: We detected 7 positive cases of m-ATTR, distributed among 3 families (Glu74Gln, Val142Ile in heterozygosity and Val142Ile in homozygosity), and 3 cases of nonpathogenic variants. CONCLUSIONS: Hereditary ATTR is a rare disease but is present in nonendemic areas and should therefore be considered in the differential diagnosis of patients with polyneuropathy and/or heart failure with preserved ejection fraction.

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.

L-arabinose resistance test with Salmonella typhimurium as a primary tool for carcinogen screening.

The L-arabinose resistance test with Salmonella typhimurium (Ara test) is a forward mutation assay which selects a single phenotypic change (from L-arabinose sensitivity to L-arabinose resistance) in a unique tester strain (an araD mutant). The present study examined the ability of the Ara test to identify as mutagens different classes of chemical carcinogens, including six with detection problems in most bacterial assays. A noncarcinogen related in chemical structure to the selected carcinogen was included whenever possible. A total of 25 organic compounds was assayed by means of a standard mutagenesis procedure: the plate-incorporation test in the presence (if required) of 10% S9 from rat liver induced with Aroclor 1254. Chemicals giving negative or inconclusive results were then retested using other common in vitro mutagenesis conditions. The Ara test detected as mutagens all but one (Urethane) of the 15 established carcinogens and six out of the nine chemicals with questionable or nonassayed carcinogenicity. The compound mutagenic at the lowest dose was ethionine (0.57 nmol), one of the carcinogens undetected by the popular histidine reverse mutation assay (Ames test) or by the SOS chromotest. Actually, only benzo(a)pyrene was found mutagenic at a dose (0.77 nmol) close to that of ethionine. The data reported in this paper suggest that the Ara forward mutation test is less prone than the His reverse mutation test or the SOS chromotest to misclassify as negative carcinogenic compounds. Consequently, it should be considered as an alternative to other bacterial assays in general, massive, and primary screening for genotoxic carcinogens.

[Esophageal atresia type I. Is impossible possible?]. / Atresia de esófago tipo I. Es posible lo imposible?

INTRODUCTION: Treatment of esophageal atresia with "long gap" remains difficult and controversial. According to the idea that esophageal anastomosis is imposible in most cases, several esophageal substitution methods have been proposed, as esophagocoloplasty, gastric transposition or reversed gastric tube. Nevertheless reconstruction of native esophagus is accepted as the best option if posible. "Long gap" definition is imprecise, expressed by variability in percent of these cases in total esophageal atresias reported in different series in literature. We report our experience in seven cases type I esophageal atresia with long gap and the different therapeutic options used, with attention to delayed or early esophageal anastomosis feasibility and outcome. MATERIAL AND METHODS: We have treated 121 patients with esophageal atresia from whom we analized 7 cases with pure esophageal atresia with "long gap" (5.8%). Six patients underwent gastrostomy and two gastrostomy and esophagostomy. Five patient underwent primary repair with esophageal anastomosis, delayed between 14 days and 4 months in 4 cases. One patient underwent esophageal anastomosis in the first day without gastrostomy. Retroesternal esophagocoloplasty was performed in 2 patients about their first year of life. Esophagogram was done in first month after surgery and pH monitoring of gastroesophageal reflux. Follow-up ranged from 6 months to 28 years. RESULTS: Esophageal anastomosis was feasible in all 5 patients in whom it was tried. Stricture occurred in two patients, one patient underwent anastomotic resection and new esophageal anastomosis. Esophageal reflux was present in two patients, one of them required funduplication. One patient was dead by complications of cardiac malformation. Remaining patients have normal swallowing and are in normal growth curves. Patients with esophagocoloplasty had not relevant early or late complications. CONCLUSIONS: In most pure esophageal atresia, delayed or even early esophageal anastomosis is feasible, making use of surgical and anesthesic sources that favour it. Esophageal substitution techniques can be reserved if this approach fails.

Genetical and biochemical characterization of QA-3 mutants and revertants in the QA gene cluster of Neurospora crassa.

The qa-3 gene, one of the four genes in the qa gene cluster, encodes quinate (shikimate) dehydrogenase (quinate: NAD oxidoreductase, ER 1.1.1.24), the first enzyme in the inducible quinic acid catabolic pathway in Neurospora crassa. Genetic analyses have localized 26 qa-3 mutants at 11 sites on the aq-3 genetic map on the basis of prototroph frequencies. Certain mutants, e.g., 336-3-10 and 336-3-3, are located at opposite ends of the qa-3 gene. Data from four-point crosses (qa-1s mutant 124 X five different qa-3 mutants in triple mutants qa-3, qa-4, qa-2) indicate the following orientation of the qa-3 gene within the qa cluster; qa-1, qa-3 mutant 336-3-10 ("left" end) qa-3 mutant 336-3-3 ("right" end), qa-4, qa-2. Ultraviolet-induced revertants have been obtained from 14 of the qa-3 mutants. The revertable mutants fall into two major classes: those that revert by changes either at the same site or at a second site within the qa-3 gene, and those that revert by unlinked suppressor mutations. The intragenic revertants can be further distinguished by quantative and/or qualitative differences in their quinate dehydrogenase activities. Some revertants with activities either equivalent to or less than wild type produce a thermostable enzyme, and others an enzyme which is thermolabile in vitro at 35 degrees. A concentration of quinic acid or shikimic acid as low as 50 micron protects the enzyme markedly from heat inactivation. The genetic organization and the orientation of the qa-3 gene are discussed with respect to its direction of transcription and to the possible localization of a promoter (initiator) region(s) within the qa gene cluster.

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.

Mutagenesis in Escherichia coli lacking catalase.

Escherichia coli K-12 strains completely lacking catalase activity due to mutations in katG, katE, and katF genes were constructed in order to assess the role of hydrogen peroxide in mutagenesis. Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance. Lethality was measured at experimental conditions equivalent to those of the mutant yield by using a mixed culture of pairs of isogenic strains distinguished by their differential nutritional requirements. Deficiency in katG, katE, and katF genes leads to an enhanced spontaneous mutation rate as well as an enhanced sensitivity to both the lethal and mutagenic effects of hydrogen peroxide or an H2O2-generating mixture of compounds, such as coffee. To compare further the responses of the catalase-deficient bacteria to those of catalase-proficient counterparts, other genotoxins were analyzed. Both catalase-deficient and catalase-proficient strains were equally mutated by MMS, 4-NQO, and ultraviolet light. It is concluded that the bacterial strains and the mutagenicity tests described in the paper represent a useful tool to study the role of H2O2 in mutagenesis.

Role of classical nitroreductase and O-acetyltransferase on the mutagenicity of nifurtimox and eight derivatives in Salmonella typhimurium.

This study investigates the mutagenicity of nifurtimox (NFX) and eight analogues in Salmonella typhimurium indicator strains that possess different levels of classical nitroreductase or O-acetyltransferase activities. The NFX analogues tested replace the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1G); pyrazol-1-yl (1B); benzimidazol-1-yl (1E); 1,2,4-triazol-4-yl (1D); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (1I); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (1H); 1-adamantyl (ADA); and 4,6-diphenylpyridin-1-yl-2-one (1K). In the genetic backgrounds of the standard Ames tester strains TA98 and TA100, these bacteria combine the L-arabinose resistance forward mutation assay (Ara test) with a deficiency or overproduction of either nitroreduction or O-acetylation. The Ara test revealed, in agreement with previous findings, important differences between TA98 and TA100 and demonstrated, moreover, that these genetic differences are of significance in mutagenicity testing with nitrofuran compounds. The Ara test also indicated dissimilarities between the metabolic activation of NFX and its analogues, these compounds being classified in three different groups according to their mutagenicity toward strain BA14 (genetic background of TA98) and its derivatives. The first group included analogues (1G, 1E, 1I, and ADA) that showed similar mutagenic potency in all bacterial strains. These compounds are considered not to be substrates for both classical nitroreductase and O-acetyltransferase. The second group included compounds (analogues 1B and 1K, and the reference drug NFX) with increased mutagenicity toward the strain overproducing the classical nitroreductase, and/or reduced mutagenicity toward the corresponding deficient bacteria. These compounds are considered to be activated by the classical nitroreductase. The third group (analogues 1D and 1H) was activated by bacterial O-acetyltransferase, and consequently showed increased and decreased mutagenicity with the particular overproducer or deficient bacterial strain, as compared to their isogenic parentals. Previous reports have pointed out interest in NFX analogue 1H as a promising candidate for the replacement of NFX. The present study further enhances the putative interest of compound 1H, based on the different metabolic activation pathway exhibited by this analogue as compared to the parental drug, NFX.

The influence of DNA repair by Ogt alkyltransferase on the distribution of alkylnitrosourea-induced mutations in Escherichia coli.

To determine the influence of DNA repair by Ogt alkyltransferase on the distribution of alkylnitrosourea-induced mutations, we have analysed in Ogt-proficient and Ogt-deficient bacterial strains the DNA sequence changes of a total of 357 independent mutations occurring within the initial part of the lacl gene of Escherichia coli. The majority (>80%) of mutations induced by either N-ethyl-N nitrosourea (ENU) or N-methyl-N-nitrosourea (MNU) in the two genetic backgrounds were G:C --> A:T transitions, consistent with the predominant role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. The analysis of the distribution of G:C --> A:T transitions induced by ENU in Ogt+ and Ogt bacteria reveals an influence of the 5'-flanking base at the level of repair by Ogt alkyltransferase. The Ogt protein appears more efficient at repairing O6-ethylguanine lesions, which are flanked 5' by a G or C, in agreement with previously reported data from our group for ethylmethane sulfonate. In contrast, no preference could be inferred for the repair of O6-methylguanine lesions by Ogt protein. These results seem to indicate that the preference of the Ogt alkyltransferase to repair certain DNA sequences might be a function of the size of the alkyl group. The importance of the alkyl group length has been described also at the level of the (A)BC excinuclease machinery that seems to have a DNA sequence specificity opposite to that of Ogt alkyltransferose.

Mutagenicity testing in Salmonella typhimurium strains possessing both the His reversion and Ara forward mutation systems and different levels of classical nitroreductase or O-acetyltransferase activities.

The induction of forward mutations to L-arabinose resistance (AraR) and of reversions to histidine prototrophy (His+) can be quantitatively compared in Salmonella typhimurium BA strains. The BA bacteria carry the araD531 allele required for the Ara assay and a his auxotrophy (hisD3052 or hisG46) required for the His assay. In this study, 2 new sets of BA indicator strains have been constructed in order to combine the Ara forward and the His reverse mutation assays of S. typhimurium with deficiency, or over-production, in either classical nitroreductase or O-acetyltransferase for mutagenicity testing of nitro-containing chemicals. Nine mutagens with different chemical structures were tested to compare the specific mutagenic sensitivities of the new constructions with those of the parental and of the conventional TA indicator bacteria. The Ara test, which responded with high sensitivity to all chemicals tested, revealed important differences between the standard tester strains TA98 and TA100 with respect to the activation of mutagens considered to be dependent on classical nitroreductase activity. Total correspondence was found between the specific mutagenic sensitivities of the defective and the overproducing bacteria in the genetic background of TA98 but not in that of TA100. In the genetic background of TA100, chemicals such as nitrofurantoin and nitrofurazone displayed 10-fold reduced mutagenicity to the "classical nitroreductase" defective strain without increasing mutagenicity to the corresponding overproducing bacteria. This discrepancy might be attributed to the greater nitroreduction capability of strain TA100 (68.12 nmole/min/mg protein) as compared to TA98 (24.42 nmole/min/mg protein), by assuming that nitrofurantoin and nitrofurazone are such good substrates for classical nitroreductase that the additional enzyme activity produced from the corresponding overexpressing plasmid when present in TA100 no longer affected their metabolic activation. We propose that the Ara forward mutation test carried out in the set of over-producing bacteria constructed in the genetic background of TA98 might play a role for routine testing of large number of samples. The isogenic defective strains could be used in cases of uncertain results with the corresponding over-producing bacteria. Finally, the reversion of the his alleles accompanying the Ara assay in the BA strains could play a role in assessing the presence of mixtures of chemicals with different mutagenic specificity in samples of environmental relevance such as urban air, foods, and water.

Direct-acting mutagenic activity in white, rosé, and red wines with the Ara test of Salmonella typhimurium.

Thirty-two commercially produced white, rosé, and red wines from Spain were assayed for genotoxicity. The Ara forward mutagenicity assay with Salmonella typhimurium served as the test system. All the wines were mutagenic in the absence of mammalian microsomal activation (S9 mix) and/or glycosidase activities with the exception of one rosé wine which gave a clear dose-response relationship, although its mutagenic potency was considered statistically nonsignificant. The mutagenic activity covered nearly a 30-fold range. Compared to white and rosé wines, red wines showed the highest levels of mutagenicity; this wine type included four "very potent" (greater than 3,000 AraR mutants/ml) mutagenic wines. The level of wine mutagenicity did not correlate with either the region or the year of production (vintage). Individual winery methods are suggested as primarily responsible for variations in mutagenic activity. The present study with the Ara test supports the possibility that wine components other than the flavonols quercetin and rutin are the major putative mutagens: (1) white wines, as well as rosé or red wines, were detected as being mutagenic; (2) in no case was activation required for the detection of mutagenicity; (3) mutagen(s) were detected mainly (red wine) when not exclusively (white and rosé wine) in the polar fraction from XAD-2 chromatography. The high sensitivity of the Ara test has allowed the screening of the mutagenicity of a variety of wines with no previous process of extraction or concentration. The comparison of the mutagenic activity of the entire complex mixture to that of its lyophilized residue has revealed a positive synergistic role for ethanol in the mutagenicity of certain wines. Finally, this work suggests that the Ara test is a useful tool for mutagenicity screening in wines. Thus, this test might play an important role in elucidating the genotoxic mechanism of action of alcoholic beverages, and for studying optional production methods to decrease the mutagenicity of commercial wines.

Influence of DNA repair by (A)BC excinuclease and Ogt alkyltransferase on the distribution of mutations induced by n-propyl-N-nitrosourea in Escherichia coli.

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increment in mutation induction by N-propyl-N-nitrosourea (PNU). Irrespective of the presence or the absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM PNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Some increased susceptibility to mutagenesis by PNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM PNU within the initial part of the lacl gene were recovered from Uvr+ Ogt-, Uvr- Ogt+, and Uvr- Ogt- bacteria. A total of 439 independent mutations were characterized by DNA sequence analysis. The PNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base and DNA-strand associated with the PNU-induced G:C-->A:T transitions. Preferences different from those previously reported for the ethylating (ENU) and methylating (MNU) analogs were detected. We indicate that these differences might be caused by the PNU possibility of giving iso-propyl adducts, in addition to the expected n-propyl adducts, and by possible preferences in the initial distribution of these lesions as well as in their repair by the (A)BC excinuclease and the Ogt ATase of E. coli.

Oxidative mutagenesis in Escherichia coli strains lacking ROS-scavenging enzymes and/or 8-oxoguanine defenses.

Escherichia coli strains with different combinations of null mutations in the katG, katE, sodA, sodB, fpg, and mutY genes were constructed to compare their spontaneous mutation frequencies and sensitivities to various oxidants with those of bacteria solely deficient in catalase (katG katE) or cytosolic superoxide dismutase (sodA sodB) and the parental strain possessing a full complement of these enzymes. The MutY DNA glycosylase represented the major protection against the mutagenic consequences of processes associated with normal aerobic metabolism. Spontaneous mutagenesis in MutY-lacking bacteria was not influenced by the absence of (A)BC excinuclease or the presence of MucAB proteins, a result consistent with 8-oxoguanine being a principal premutational lesion. In contrast, catalase and SOD represented the major protection against the genotoxic consequences of bursts of oxidative stress caused by reactive-oxygen-generating compounds. Therefore, only bacteria simultaneously defective in both katG and katE or sodA and sodB genes were hypersensitive with respect to mutability by peroxide and superoxide, respectively. These data suggest that oxidative lesions other than 8-oxoguanine contribute to mutagenesis by hydrogen peroxide and redox-cycling chemicals.

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.

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.