New aspects in deriving health-based guidance values for bromate in swimming pool water.

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1-5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460-1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose-response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587-594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73-98%) by swallowing water, followed by the dermal route (2-27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Critical evaluation of human health risks due to hydraulic fracturing in natural gas and petroleum production.

The use of hydraulic fracturing (HF) to extract oil and natural gas has increased, along with intensive discussions on the associated risks to human health. Three technical processes should be differentiated when evaluating human health risks, namely (1) drilling of the borehole, (2) hydraulic stimulation, and (3) gas or oil production. During the drilling phase, emissions such as NOx, NMVOCs (non-methane volatile organic compounds) as precursors for tropospheric ozone formation, and SOx have been shown to be higher compared to the subsequent phases. In relation to hydraulic stimulation, the toxicity of frac fluids is of relevance. More than 1100 compounds have been identified as components. A trend is to use fewer, less hazardous and more biodegradable substances; however, the use of hydrocarbons, such as kerosene and diesel, is still allowed in the USA. Methane in drinking water is of low toxicological relevance but may indicate inadequate integrity of the gas well. There is a great concern regarding the contamination of ground- and surface water during the production phase. Water that flows to the surface from oil and gas wells, so-called 'produced water', represents a mixture of flow-back, the injected frac fluid returning to the surface, and the reservoir water present in natural oil and gas deposits. Among numerous hazardous compounds, produced water may contain bromide, arsenic, strontium, mercury, barium, radioactive isotopes and organic compounds, particularly benzene, toluene, ethylbenzene and xylenes (BTEX). The sewage outflow, even from specialized treatment plants, may still contain critical concentrations of barium, strontium and arsenic. Evidence suggests that the quality of groundwater and surface water may be compromised by disposal of produced water. Particularly critical is the use of produced water for watering of agricultural areas, where persistent compounds may accumulate. Air contamination can occur as a result of several HF-associated activities. In addition to BTEX, 20 HF-associated air contaminants are group 1A or 1B carcinogens according to the IARC. In the U.S., oil and gas production (including conventional production) represents the second largest source of anthropogenic methane emissions. High-quality epidemiological studies are required, especially in light of recent observations of an association between childhood leukemia and multiple myeloma in the neighborhood of oil and gas production sites. In conclusion, (1) strong evidence supports the conclusion that frac fluids can lead to local environmental contamination; (2) while changes in the chemical composition of soil, water and air are likely to occur, the increased levels are still often below threshold values for safety; (3) point source pollution due to poor maintenance of wells and pipelines can be monitored and remedied; (4) risk assessment should be based on both hazard and exposure evaluation; (5) while the concentrations of frac fluid chemicals are low, some are known carcinogens; therefore, thorough, well-designed studies are needed to assess the risk to human health with high certainty; (6) HF can represent a health risk via long-lasting contamination of soil and water, when strict safety measures are not rigorously applied.

Review of the Ecotoxicological Properties of the Methylenedianiline Substances.

Concerning chronic toxicity, D. magna is the most sensitive species tested against MDA aquatic exposures, with a 21 days-NOEC of 0.00525 mg/L. Exposure of daphnids takes place via the aquatic phase. Other species of the same phylum (Arthropoda) appear to be less sensitive albeit with exposures via soil or sediment, with a 28 days-NOEC of 562 mg/kg d. w. soil (F. candida) and 41.3 mg/kg d. w. sediment (Hyalella azteca), for reproductive and survival endpoints, respectively. Also for acute toxicity, D. magna is more sensitive than the other species, with an 48 h-EC50 that spreads over two orders of magnitude, ranging from 0.019 to 2.7 mg/L. Fish show a more uniform reaction to MDA, with 96 h-LC50 ranging from about 20 to 60 mg/L; chronic data for fish are not available. Acute toxicity data for algae and cyanobacteria are in the range of 1-10 mg/L; based on growth rate, the 72 h-NOECr or ErC10 of MDA to algae is 0.3-9.3 mg/L.For sediment organisms, the black worm L. variegatus shows the highest sensitivity against MDA with a NOEC between ≤3.75 mg/kg and 30 mg/kg d. w., followed by the amphipod H. azteca. The lower sensitivity of L. variegatus in the second study compared to the first study is obviously attributable to the different feeding regimes (semi-continuous feeding against pre-spiked sediment). One argument might be that semi-continuous feeding allows the organisms to avoid the contaminated food. However, a change from semi-continuous feeding to sediment pre-spiked with nettle powder (Urtica sp.) results in an earlier and much stronger increase in ammonia concentration in the system. This became apparent after both studies on the blackworm were finalized. The ammonia 96 h-EC50 for the blackworm is 0.69 mg/L at pH = 8.2, and the 96 h-EC10 at pH = 8.2 is 0.33 mg/L (Hickey and Vickers, Arch Environ Contam Toxicol 26:292-298, 1994). As a result, the lower NOEC and LOEC in the second study with L. variegatus are probably attributable to interference by ammonia.MDA binds irreversibly to soil and sediment which may explain the general, but not uniform lower sensitivity of soil and sediment organisms against aquatic organisms. However, species with intense soil or sediment contact (L. variegatus and E. fetida) show in general lower NOEC values than those organisms with less direct contact (3.75 and 11.2 mg/kg d. w., respectively). On the one hand it may be hypothesized that this intense contact to soil bound MDA is one reason for the higher sensitivity; on the other hand, metabolic capacity against MDA of the organisms tested is unknown at this point in time and might as well explain differences in species sensitivity. For plants there are only acute data available, and in respect to acute toxicity L. sativa is more sensitive to MDA than E. fetida.Limited aquatic data available so far do not indicate that the toxicity of pMDA is different to MDA. In addition, the limited set of data generated with the marine M. macrocopa (crustacean), N. fustulum (diatom) and V. fisheri (bacteria) do not indicate that sea water organisms are more sensitive to MDA than fresh water organisms.In mammals, MDA is unlikely to interact with the endocrine sexual system; interaction with the adrenergic system cannot be ruled out, and effects of MDA on the thyroid hormone system have been demonstrated. MDA inhibits the thyroid peroxidase which might contribute to the thyroid gland tumors observed in chronic studies with rats and mice. Some anti-androgenic activity in in vitro studies with yeast cell did not prevail in in vivo studies with rats and mice.

The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90.

BACKGROUND: Epothilones are produced by the myxobacterium Sorangium cellulosum So ce90, and, like paclitaxel (Taxol((R))), they inhibit microtubule depolymerisation and arrest the cell cycle at the G2-M phase. They are effective against P-glycoprotein-expressing multiple-drug-resistant tumor cell lines and are more water soluble than paclitaxel. The total synthesis of epothilones has been achieved, but has not provided an economically viable alternative to fermentation. We set out to clone, sequence and analyze the gene cluster responsible for the biosynthesis of the epothilones in S. cellulosum So ce90. RESULTS: A cluster of 22 open reading frames spanning 68,750 base pairs of the S. cellulosum So ce90 genome has been sequenced and found to encode nine modules of a polyketide synthase (PKS), one module of a nonribosomal peptide synthetase (NRPS), a cytochrome P450, and two putative antibiotic transport proteins. Disruptions in the genes encoding the PKS abolished epothilone production. The first PKS module and the NRPS module are proposed to co-operate in forming the thiazole heterocycle of epothilone from an acetate and a cysteine by condensation, cyclodehydration and subsequent dehydrogenation. The remaining eight PKS modules are responsible for the elaboration of the rest of the epothilone carbon skeleton. CONCLUSIONS: The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts.

Cloning and expression of two genes of Streptomyces pilosus involved in the biosynthesis of the siderophore desferrioxamine B.

Genes involved in biosynthesis of the siderophore desferrioxamine B have been cloned from Streptomyces pilosus using a self-cloning approach with the low-copy plasmid pIJ943. Mutants in two steps of the pathway can be complemented by a 4.5-kb DNA fragment. One of these mutants is defective for the first step, lysine decarboxylation. Expression of lysine decarboxylase by the cloned DNA was found to be regulated by iron. S1 mapping and Northern blotting experiments show that iron regulation of desferrioxamine B production is exerted at the transcriptional level, and also that genes involved specify a long message, which determines at least two steps in the biosynthetic pathway.

Cloning and sequence analysis of the Corynebacterium diphtheriae dtxR homologue from Streptomyces lividans and S. pilosus encoding a putative iron repressor protein.

The iron-regulated promoter involved in desferrioxamine B synthesis of Streptomyces pilosus contains a region homologous to the iron repressor (DtxR)-binding site of the diphtheria toxin gene promoter in Corynebacterium diphtheriae [Günter et al., J. Bacteriol. 175 (1993) 3295-3302]. Here, we report the cloning and sequencing of the putative Streptomyces iron repressor gene, homologous to dtxR of C. diphtheriae. The N-terminal 139 amino acids of the deduced protein are 73% identical to DtxR.

Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei.

The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E. coli and completely sequenced. The DNA encodes five closely packed, very large open reading frames reading in one direction. As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each. The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and beta-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.

Transformation of rifamycin S into rifamycins B and L. A revision of the current biosynthetic hypothesis.

The transformation of rifamycin S into rifamycins B and L was reinvestigated in order to establish more detailed pathways. Our results exclude rifamycin O as a common progenitor in the biosyntheses of rifamycins B and L. Rifamycins B and L are formed from rifamycin S (SV) by different pathways using different C3-precursors for the biosynthesis of their glycolic acid moieties. A thiamine-dependent enzyme (decarboxylase) seems to be involved in the transformation reaction.

New rifamycins produced by a recombinant strain of Nocardia mediterranei.

A recombinant strain of Nocardia mediterranei was found to produce a number of new rifamycins which are structurally related to rifamycin S, rifamycin W and rifamycin G. This strain was derived from two Nocardia mediterranei mutants by intraspecific recombination.

3-Hydroxyrifamycin S and further novel ansamycins from a recombinant strain R-21 of Nocardia mediterranei.

The structures of 3-hydroxyrifamycin S and six further novel ansamycins isolated from the recombinant strain R-21 of Nocardia mediterranei were identified by spectroscopic methods. Three types of structure were distinguished: Type 1: Ansamycins of the rifamycin S type Type 2: Ansamycins of the rifamycin G type Type 3: Ansamycins of the rifamycin W type.

Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome.

Recombinant vectors derived from the broad-host-range mobilizable plasmid pSUP2021 were constructed and transferred by IncP-mediated conjugation from Escherichia coli to Sorangium cellulosum, where they were integrated into the chromosome by homologous recombination and maintained stably. This appears to be the first system of gene transfer to S. cellulosum.

Characterization of an iron-regulated promoter involved in desferrioxamine B synthesis in Streptomyces pilosus: repressor-binding site and homology to the diphtheria toxin gene promoter.

Desferrioxamine B is the main siderophore of Streptomyces pilosus. Its production is induced in response to iron limitation. Two genes involved in desferrioxamine production have been cloned and were found to be translated from a polycistronic mRNA that is produced only under conditions of iron limitation (T. Schupp, C. Toupet, and M. Divers, Gene 64:179-188, 1988). Here we report the nucleotide sequence of the desferrioxamine (des) operon promoter region. The transcriptional start site was localized by S1 nuclease mapping. Deletion analysis defined a 71-bp region downstream of the -35 region that is sufficient for iron regulation in the original host, S. pilosus, and also in Streptomyces lividans. Site-directed mutagenesis was used to create a mutation that abolishes iron repression. Two iron-independent mutants were obtained by deletion of part of a 19-bp region with dyad symmetry which overlaps the -10 promoter region and the transcriptional start site. The putative repressor-binding site identified by these constitutive mutations is not homologous to the consensus binding site of the Escherichia coli central iron repressor, Fur (ferric uptake regulation), but is similar to the DtxR-binding site in the iron-regulated promoter of the corynebacterial diphtheria toxin gene.

Management of pregnancy in a university hospital: a 6-year study.

In order to evaluate the obstetric care in the Obstetric Clinic of the Gynaecology and Obstetrics Department of University of Sao Paulo, the authors present a survey of the management of pregnancy during the 6-year period from 1993 to 1998. The number of deliveries increased during the study by 45% over the 6 years. During this same period the number of fetal deaths was 526 (4.48%), but there was a significant decrease (p < 0.05) in the incidence of fetal death. However, there was no concomitant increase in the proportion of pregnant women with prenatal care that could explain this improvement. Incidence of premature labor also decreased considerably. The authors believe that the increment in the number of deliveries was due mainly to the increasing number of pregnant women referred to our service. The efforts made by the service towards decreasing the time of hospitalization of both newborns in the nursery and the mothers in the hospital made this possible. Despite the increasing number of deliveries, there was a significant improvement in the management of pregnancy during the period of study. This improvement may be a consequence of the standardization of a protocol of management of pregnancy based on the recent progress in scientific and technological knowledge.

Genetic recombination in Nocardia mediterranei.

A system of genetic recombination in Nocardia mediterranei ATCC 13685 is described. This strain produces a mixture of several rifamycin antibiotics. Using haploid recombinant selection and analysis procedures similar to those applied to Streptomyces coelicolor A3(2), 14 auxotrophic markers and 1 streptomycin resistance marker were located on a circular linkage map. The linkage map of N. mediterranei seems to be similar to that of S. coelicolor A3(2).

Expression of a neomycin phosphotransferase gene from Streptomyces fradiae in Escherichia coli after interplasmidic recombination.

Plasmid pIJ2 carrying the neomycin phosphotransferase gene of Streptomyces fradiae was fused to E. coli plasmid pBR325 and the hybrid molecules were introduced into E. coli K12 by transformation. The neomycin phosphotransferase gene of the hybrid plasmid was not expressed in E. coli, except after interplasmidic recombination. Physical analysis of such an in vivo recombinant plasmid revealed that the recombination brought one neomycin phosphotransferase gene to a position downstream from the tet-promoter of pBR325. Subcloning experiments indicated that this is the gene copy expressed, and that transcription is initiated at the tet-promoter of pBR325.

A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.