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.

Gut-gonad crosstalk in mice exposed to a "chemical cocktail" combining metabolomics and microbial profile by amplicon sequencing.

Testes are very prone to be damaged by environmental pollutants, but there is a lack of information about the impact of "chemical cocktails" (CC) on the testicular metabolome and the possible influence in the gut-gonad crosstalk. For this, BALB/c mice were given flumequine and diclofenac orally in food and potentially toxic trace elements (Cd, Hg, As) in drinking water. A mice group was supplemented with selenium, a well-known antagonist against many pollutants. Our results revealed that the steroid 5-alpha-androstan-17-beta-ol propionate, suggested as a parameter of androgenicity independent of testosterone levels, proline that improves reproductive indicators in male rabbits affected by environmental stress) among others metabolites are only present after CC exposure with rodent and selenium supplemented diet. Selenium also antagonized the up-or down-regulation of anandamide (20:l, n-9) (p < 0.001 and FC 0.54 of CC vs C but p > 0,05 and FC 0.74 of CC-Se vs C), that regulates gonadotropin-releasing hormones in mammals, 2,3-dinor-11b-PGF2a (p < 0.001 and FC 0.12 of CC vs C but p > 0,05 and FC 0.34 of CC-Se vs C), which has been related with reproductive hormones, besides others testicular metabolites altered by the exposure to the CC and reversed the levels to control. Moreover, numerous significant associations between gut microbes and testicular metabolites indicated a possible impact of pollutants in the testes mediated by gut microbiota due to a gut-gonad crosstalk.

Impact of "chemical cocktails" exposure in shaping mice gut microbiota and the role of selenium supplementation combining metallomics, metabolomics, and metataxonomics.

Biological systems are exposed to a complex environment in which pollutants can interact through synergistic or antagonistic mechanisms, but limited information is available on the combined effects. To this end, conventional and antibiotic-treated (Abx) mice models were fed regular rodent or selenium (Se) supplemented diets and exposed to a "chemical cocktail" (CC) including metals and pharmaceuticals. Metallomics, metabolomics, and metataxomics were combined to delve into the impact on gut microbiota, plasma selenoproteome, metabolome, and arsenic metabolization. At the molecular level, Se decreased the concentration of the antioxidant glutathione peroxidase in plasma and increased the arsenic methylation rate, possibly favoring its excretion, but not in the Abx and also plasma metabolomes of Abx, and Abx-Se were not differentiated. Moreover, numerous associations were obtained between plasma selenoproteins and gut microbes. Se-supplementation partially antagonizes the gut microbiota alteration caused by Abx, and slightly by CC, but strongly altered profiles were observed in CC-Abx-Se, suggesting synergistic deleterious effects between pollutants, Abx and Se. Moreover, although CC and Abx changed gut microbiota, several common taxa were enriched in CC-Abx and control mice, indicating possible synergistic effects. Our results suggest a potential beneficial impact of supplementation, but mediated by gut microbes being reversed in their absence.

Mice brain metabolomics after the exposure to a "chemical cocktail" and selenium supplementation through the gut-brain axis.

Several environmental pollutants have been shown to damage brain and affect gut microbiota. Limited evidence is available about the impact of "chemical cocktails" (CC) of xenobiotics on brain metabolome and their possible influence in the gut-brain crosstalk. To this end, BALB/c mice were exposed to heavy metals (As, Hg, Cd) and pharmaceuticals (diclofenac and flumequine) under regular rodent diet or supplemented with selenium (Se). Selenium, an antioxidant well-known for its antagonism against the neurotoxicity of several pollutants, modulated several brain metabolic impairments caused by CC (e.g., brain levels of the excitatory amino acid N-acetyl aspartic acid) by influencing mainly the metabolisms of purine, glycosylate and dicarboxylate, glutamate, glycerophospholipid, alanine and aspartate. Numerous associations were obtained between brain metabolites and gut microbes and they changed after Se-supplementation (e.g., Lactobacillus was positively associated with a brain ceramide, phosphoserine, phosphocholine, vitamin D3 derivative, fatty acids, malic acid, amino acids, and urea after the exposure, but not after Se-supplementation). Our results showed numerous evidences about the impact of CC on brain metabolome, the potential role of Se as an antagonist and their impact on the gut-brain axis. Further research is needed to understand the complex mechanism of action implied on CC-brain-microbiota interactions.

Targeted and untargeted metabolomic analysis of Procambarus clarkii exposed to a "chemical cocktail" of heavy metals and diclofenac.

Water pollution poses an important problem, but limited information is available about the joined effects of xenobiotics of different chemical groups to evaluate the real biological response. Procambarus clarkii (P. clarkii) has been demonstrated to be a good bioindicator for assessing the quality of aquatic ecosystems. In this work, we studied the bioaccumulation of cadmium (Cd), arsenic (As) and diclofenac (DCF) in different tissues of P. clarkii during 21 days after the exposure to a "chemical cocktail" of As, Cd and DCF, and until 28 days considering a depuration period. In addition, a combined untargeted and targeted metabolomic analysis was carried out to delve the metabolic impairments caused as well as the metabolization of DCF. Our results indicate that As and Cd were mainly accumulated in the hepatopancreas followed by gills and finally abdominal muscle. As and Cd show a general trend to increase the concentration throughout the exposure experience, while a decrease in the concentration of these elements is observed after 7 days of the depuration process. This is also the case in the abdominal muscle for Cd, but not for As and DCF, which increased the concentration in this tissue in the depuration phase. The hepatopancreas showed the greatest number of metabolic pathways affected. Thus, we observed a crucial bioaccumulation of xenobiotics and impairments of metabolites in different tissues. This is the first study combining the exposure to metals and pharmaceutically active compounds in P. clarkii by untargeted metabolomics including the biotransformation of DCF.

The new frontier of bone formation: a breakthrough in postmenopausal osteoporosis?

OBJECTIVE: Osteoporosis is a chronic disease that accelerates after menopause in many women. Most of the pharmacologic attempts to control the disease, such as hormone therapy, have emphasized the constraint of bone resorption. Since recent years have witnessed important advances in the field of bone formation, this review aims to update the present knowledge on the mechanisms affecting osteoblastogenesis and on the therapeutic results achieved by recently approved drugs. METHOD: We sought peer-reviewed, full-length basic and clinical articles published between 1995 and May 2008 using a PubMed search strategy, with the terms osteoporosis and osteoblast, osteoporosis and strontium ranelate, and osteoporosis and parathyroid hormone (PTH). This search was further supplemented by a hand-search of reference lists of selected review papers. After crossing-cleaning the reference lists, some 800 articles were selected. Articles on regulators of osteoblast differentiation and function, together with well-designed clinical studies, were surveyed. RESULTS: A complex network of systemic and local factors regulates osteoblastogenesis. Advances in fracture protection have been published in clinical studies with PTH. Some investigators claim an anabolic effect for strontium ranelate, which also confers protection against fracture. CONCLUSION: The control of bone formation offers new clinical potential. Stimulation of bone formation by PTH has translated into fracture protection. The action of strontium ranelate has been claimed to be mediated by some level of bone formation, but this hypothesis still needs clarification.

Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica.

The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in hepatic lymph nodes (HLN) and livers from sheep infected with Fasciola hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-ß, TNF-α and IL-1ß genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1ß during chronic stages of infection with F. hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. hepatica vaccinated and infected sheep.

Th1/Th2 balance in the liver and hepatic lymph nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola hepatica.

The expression of IFNγ and IL4 was quantified using q-PCR in the liver and hepatic lymph nodes (HLN) of sheep during early stages of infection with Fasciola hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.

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.

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.

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.

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.

Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12.

In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents. The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies. By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs. methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase). Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein. These results support the reported in vitro substrate specificities for both ogt and ada ATases. The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase. Deficient bacterial derivatives showed, by contrast, linear mutation induction responses. The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10). The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives. This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected. In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)

The effectiveness of the O(6)-alkylguanine-DNA alkyltransferase encoded by the ogt(ST) gene from S. typhimurium in protection against alkylating drugs, resistance to O(6)-benzylguanine and sensitisation to dibromoalkane genotoxicity.

Here we demonstrate that the Ogt(ST) from Salmonella typhimurium is a highly efficient O(6)-alkylguanine-DNA alkyltransferase (AGT) in affording protection against antitumour chloroethylating drugs (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)). In addition, Ogt(ST) is refractory to O(6)-benzylguanine (BG) inactivation and its expression provides only minor sensitisation to genotoxicity by environmental dibromoalkanes (DBE). No other of the assayed bacterial or human AGTs displayed such advantageous properties for chemoprotective gene therapy strategy. Our observations indicate that the Ogt(ST) AGT might be, under some circumstances, of potential use to improve cancer chemotherapy. At least, its properties may provide further insight into the design of human AGT variants that could be expressed in normal or tumour cells to provide either protection or ablation.

Biochemical and genetic indices of marine pollution in Spanish littoral.

Increased activities of several detoxifying and antioxidant enzymes were detected in mollusc and fish from Spanish littoral areas with high metal contents. Ethanolic extracts from molluscs contained direct-acting and polar genotoxins of oxidative type, which were detected by strain TA102 of S. typhimurium and catalase-deficient strains of E. coli. Animals from contaminated sites contained less genotoxins than those from control areas. Polluted fishes displayed highly induced cytochrome P-450 activity and increased promutagen activation capabilities. In addition, specific forms of glutathione transferase and superoxide dismutase were induced, particularly highly acidic forms.

Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains isolated from invasive and non-invasive infections from Mexico and the USA during 1999-2010.

OBJECTIVE: To compare the prevalence, phenotypes, and genes responsible for erythromycin resistance among Streptococcus pyogenes isolates from Mexico and the USA. METHODS: Eighty-nine invasive and 378 non-invasive isolates from Mexico, plus 148 invasive, 21 non-invasive, and five unclassified isolates from the USA were studied. Susceptibilities to penicillin, erythromycin, clindamycin, ceftriaxone, and vancomycin were evaluated according to Clinical and Laboratory Standards Institute (CLSI) standards. Phenotypes of erythromycin resistance were identified by triple disk test, and screening for mefA, ermTR, and ermB genes was carried out by PCR. RESULTS: All isolates were susceptible to penicillin, ceftriaxone, and vancomycin. Erythromycin resistance was found in 4.9% of Mexican strains and 5.2% of USA strains. Phenotypes in Mexican strains were 95% M and 5% cMLS; in strains from the USA, phenotypes were 33.3% iMLS, 33.3% iMLS-D, and 33.3% M. Erythromycin resistance genes in strains from Mexico were mefA (95%) and ermB (5%); USA strains harbored ermTR (56%), mefA (33%), and none (11%). In Mexico, all erythromycin-resistant strains were non-invasive, whereas 89% of strains from the USA were invasive. CONCLUSIONS: Erythromycin resistance continues to exist at low levels in both Mexico and the USA, although the genetic mechanisms responsible differ between the two nations. These genetic differences may be related to the invasive character of the S. pyogenes isolated.

DNA repair by Ogt alkyltransferase influences EMS mutational specificity.

Forward mutations induced by ethylmethane sulfonate (EMS) in the lacI gene of Escherichia coli were recovered from bacteria proficient or deficient in the alkyltransferase encoded by the constitutive ogt gene. EMS doses of 100 or 200 mM (Ogt+) and of 50 mM (Ogt-) were selected from the corresponding dose-response curves for DNA sequence analysis. A total of 239 induced mutations affecting the N-terminal region of the lacI gene were characterized. All mutations were G:C-->A:T transitions, consistent with the predominant role of the O6-ethylguanine miscoding lesion in mutagenesis by EMS. In the Ogt+ spectrum at the lowest tested dose of 100 mM EMS, guanines preceded by an A or T base at the 5' side were on average 3.2 times more likely to mutate than those preceded by a G or C base. This bias diminished at the higher EMS dose (200 mM) and disappeared in the Ogt- genetic background. Previously reported data for Ogt+ bacteria in a Uvr-proficient background show an opposite bias in favor of mutations at guanines preceded by a G or C base. The overall 5' flanking base influence was estimated as 8-fold. These data suggest that DNA repair by Ogt alkyltransferase plays an important role in the processing of ethylation-induced lesions responsible for GC-->AT transitions, influencing their ultimate distribution with respect to sequence context. The data further suggest that Ogt and UvrABC excision repair, the two major mechanisms of protection against the biological consequences of long-chain alkylating agents, show different DNA sequence specificity and that the relative importance of these two systems is highly dependent upon the chemical dose.

DNA sequence analysis of spontaneous lacI-d mutations in O6-alkylguanine-DNA alkyltransferase-proficient and -deficient Escherichia coli.

Spontaneous mutagenesis in O6-alkylguanine-DNA alkyltransferase-proficient and -deficient (ada ogt mutants) Escherichia coli was studied in two ways: in bacteria growing in nonselective liquid medium and in bacteria resting on selective agar plates. ATase mutants showed similar spontaneous mutation rates as ATase proficient bacteria during growth phase; an excess of mutants arising in nondividing cells. The resting-associated mutagenesis in ada + ogt + uvr- bacteria was biphasic; the high sensitive range being triggered beyond the first 6 days after plating. Contrarily, spontaneous Lacc mutants from ada- ogt- uvr- cells steadily increased over the 8 day period of plate incubation. These results suggested that, in the absence of nucleotide excision repair, the repair by both the Ada and the Ogt ATases is not saturated until the cells have been resting for 6 days. The spontaneous LacI-d mutation spectrum of ada + ogt + uvr- bacteria growing in non-selective liquid medium served as a baseline to determine the mutation events increased in the ATase-deficient derivative upon prolonged incubation on selective plates. The percentage of G:C-->A:T transitions, presumably driven by unrepaired O6-alkylguanine lesions, was increased at the expense of other mutation types. G:C-->A:T transitions accumulated with a pronounced 5'PuG bias, suggesting that the endogenous metabolite(s) responsible for this mutation class is an SN1 type alkylating compound(s). Accordingly, the site distribution of G:C-->A:T transitions in nondividing ATase defective bacteria showed similarities with the spectra induced by alkylnitrosoureas, particularly with those generating bulky alkylated DNA adducts.

Mammalian cells expressing Escherichia coli O6-alkylguanine-DNA alkyltransferases are hypersensitive to dibromoalkanes.

The effect of expression of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, on the growth inhibitory effects of the dibromoalkanes (DBA) dibromomethane (DBM) and dibromoethane (DBE) was determined in Chinese hamster lung fibroblasts transfected with and expressing high levels of the Escherichia coli alkyltransferase (ATase) genes. These included the ogt gene and complete or truncated versions of the E. coli ada gene encoding either O6-alkylguanine (O6-alkG) or alkylphosphotriester (alkPT) ATase activities. The functional activity of the ATase in these cells was demonstrated by in vitro assay of cell extracts using 3H-methylated DNA as a substrate, and by the protection they provided against the growth inhibitory effects of methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) and the chloroethylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU). However, cells expressing the full length or the O6-alkG ATase region, but not the alkPT ATase region, of Ada were found to be more sensitive to the growth inhibitory effects of the DBA; Ogt expression sensitized cells to DBM but not significantly to DBE. Addition of DBA to cell extracts depleted O6-alkG ATase activity on the methylated DNA substrate, but had no effect on alkPT ATase activity. This suggests that ATase-mediated sensitization of the intact cells may be related to the inactivation of the ATase protein. Addition to the cell culture medium of GSH or buthionine sulfoximine in attempts to augment or deplete cellular levels of GSH had no marked effect on the ATase-mediated sensitization to DBA. This suggests that rather than GSH-mediated DNA damage, the effect may be mediated by a DNA adduct caused by the oxidative metabolic pathway. These observations indicate that expression of ATase may have a detrimental effect on cellular sensitivity to environmentally relevant alkylating agents.