Stereoselectivity in Ene Reactions with ^sup 1^O2: Matrix Effects in Polymer Supports, Photo-oxygenation of Organic Salts and Asymmetric Synthesis
The ene reaction of chiral allylic alcohols is applied as a tool for the investigation of intrapolymer effects by means of the stereoselectivity of the singlet-oxygen addition. The diastereo selectivity strongly depends on the structure of the polymer, the substrate loading degree and also on the degree of conversion demonstrating additional supramolecular effects evolving during the reaction. The efficiency and the stability of polymer-bound sensitizers were evaluated by the ene reaction of singlet oxygen with citronellol. The ene reaction with chiral ammonium salts of tiglic acid was conducted under solution phase conditions or in polystyrene beads under chiral contact ion-pair conditions. The products thus obtained precipitate during the photoreaction as ammonium salts. Moderate asymmetric induction was observed for this procedure for the first time.
Singlet oxygen is a versatile reagent in organic oxidation chemistry. Because this molecule can be generated by numerous thermal methods, singlet-oxygen chemistry is often not considered per se as a photochemical topic. The most practical method to generate singlet oxygen in solution, however, is triplet sensitization by use of electronically excited triplet states of dye molecules. Furthermore, singlet oxygen is an electronically excited molecule and can decay to its ground state by radiation-less or radiative processes in addition to chemical reactions. The chemical process that leads to the oxidation of organic substrates by transfer of both oxygen atoms is termed Type-II photo-oxygenation (1-3). The atom economy of such a process is 100%, a major advantage over other oxidants, such as hydrogen peroxide, that can only reach an atom economy of 48%, if calculated for the formation of epoxides from alkenes (4). The primary peroxidic products from Type-II photo-oxygenation can be reduced to numerous polyoxygenated derivatives either with loss of the original oxygen content (allylic alcohols from allylic hydroperoxides) or with conservation of the oxygen content (1,2-and 1,4-diols from dioxetanes and endoperoxides, respectively, or epoxy-alcohols from allylic hydroperoxides by means of titanium[IV]-catalyzed oxygen transfer).
Singlet-oxygen reaction media
In the last decade, the area of polymer-supported organic reactions (5-7) and polymer-supported catalysts (8,9) has impressively increased. This is obvious for solid-phase synthetic chemistry, where reactions are carried out in resins and/or catalyzed by supported catalysts. Photo-oxygenation in solution with the use of insoluble polymer-bound sensitizers facilitates the problem of dye recovery. The first polymer-bound sensitizer was the now commercially available polystyrene-bound Rose Bengal developed by Schaap et al. (10) followed by a series of immobilized sensitizers, e.g. the immobilized fullerene C^sub 60^ (11,12), ionic porphyrins immobilized on cationically functionalized polystyrene (13), tetrakis(4-hydroxyphenyl)porphyrin supported to polyethylene glycol (14), aluminum(III) tetracarboxyphthalocyanine bound to poly(styrene-co-chloromethylstyrene) (15), polystyrene-bound benzophenones (16), immobilized pyrylium salts on Merrifield resins (17), sensitizer-incorporated nation membranes (18), or ion-exchange resins ionically bound to photosensitizers (19). Other heterogeneous catalysts using clay (20), silica (21), and zeolites (22,23) as support materials were also recently developed. From the viewpoint of solution photochemistry, the use of nonpolar solvents enhances dye oxidation and bleeding, especially if long reaction times are needed, which decreases the singlet-oxygen quantum yield and hence the reaction efficiency. On the other hand, photo-oxygenation reactions carried out in aqueous solutions are also not favored, due to low solubility of most organic substrates, the low singlet-oxygen lifetime, and hydrophobic aggregations of nonpolar sensitizers (leading to self quenching), which as a consequence reduces the triplet lifetime (24,25), We have recently reported a solution to circumvent some of the mentioned technical problems: Under solvent-free reaction conditions, in which the substrates are embedded in a porphyrin-loaded polystyrene polymer matrix, irradiation and product isolation accompanied by complete sensitizer separation by extraction with ethanol offers a shortcut to green photo-oxygenation reactions (26).
Two major problems in photo-oxygenation chemistry appeared unsolved until recently: (a) altering the regio- and diastereoselectivity of singlet-oxygen ene or [4+2]-cycloadditions by optimizing the reaction media, and (b) making singlet oxygen chiral, i.e. introducing high enantioselectivity into singlet-oxygen ene or [4+2]-cycloaddition reactions by catalytic methods.
The singlet-oxygen ene reaction
The singlet-oxygen ene reaction was discovered in 1943 by G. O. Schenck (27,28). In the course of this reaction, ^sup 1^O^sub 2^ attacks one center of a CC double bond with abstraction of an allylic hydrogen atom and shift of the double bond (Scheme 1). As a result of this reaction, allylic hydroperoxides are formed, compounds that are versatile intermediates for the synthesis of allylic alcohols, epoxides, epoxyalcohols, 1,2-diols, and 1,2,3-triols. Several mechanisms have been postulated for this reaction, most recently the two-step no-intermediate mechanism involving a bifurcating transition state with perepoxide structure (29), as well as 1,4-biradicals (30), 1,4-zwitterions (31), perepoxide (32), dioxetane or exciplex intermediates.
Lawsuit against fertilizer maker dismissed
A lawsuit filed against an explosives company that may have produced fertilizer used in the Oklahoma City bombing has been dismissed after a judge ruled there was no legitimate claim of negligence.
The lawsuit filed against ICI Explosives USA, Inc., by attorneys representing about 250 victims and survivors of the bombing accused the company of illegally selling “explosive-grade” am-monium nitrate on the open market as fertilizer.
The lawsuit, which sought unspecified damages, also alleged the company failed to add an ingredient to the ammonium nitrate that could make it less volatile.
The company maintained that the ammonium nitrate was sold in the less volatile pellet form instead of a powder, and denied that any ingredient would render the substance inert.
In an order handed down late Tuesday, U.S. District Judge David Russell ruled the plaintiffs failed to state claims of negligence, negligent infliction of emotion distress, intentional infliction of emotional distress, manufacturers’ products liability, liability for hazardous or abnormally dangerous activity, fraud or deceit.
“Judge Russell has granted ICI Explosives USA’s motion for dismissal, confirming that ICI is in no way responsible for the tragedy in Oklahoma City,” company spokesman Neal Medick said in a prepared statement. “The judge’s ruling places the responsibility squarely where it belongs — on the terrorists.”
Willie Watson, whose grandson, P.J. Allen, was severely burned in the explosion and a plaintiff in the suit, expressed shock Wednesday at the judge’s ruling.
“That kind of upsets me,” Watson said. “They took the explosives out of it for overseas. I still feel like they were liable.”
Pure ammonium nitrate is only sold as an explosive in some foreign countries.
The Alfred P. Murrah Federal Building was destroyed April 19, 1995, with a 4,800-pound bomb made mostly of ammonium nitrate and fuel oil, authorities believe. The blast killed 168 people.
The men accused of the bombing, Timothy McVeigh and Terry Nichols, are believed to have purchased ammonium nitrate at the Mid-Kansas Cooperative in McPherson, Kan., which is supplied by ICI Explosives.
The lawsuit was filed by attorneys John Merritt of Oklahoma City and Johnnie Cochran, who is better known for successfully defending O.J. Simpson. Merritt was in a trial in Missouri and unavailable for comment.
McVeigh and Nichols face federal murder and conspiracy charges in the bombing.
If convicted, they could be sentenced to death.
Don’t put the SCR cart before the CO horse
The excellent and informative article on the upcoming HRSG User’s Group guidelines suggests the placement of the CO catalyst after a selective catalytic reduction (SCR) NOx catalyst, presumably in a lower temperature region. Although potentially less SO2 to SO3 conversion would occur [with this arrangement], the excessive ammonia slip would cause additional NOx to be generated in the CO catalyst, which is undesirable.
Many CO catalysts are placed in a higher temperature region due to increased effectiveness for a given amount of catalyst volume–although gas-turbine backpressure will increase. The negative result is that more SO2 will be converted to SO3, in some cases by as much as 80%, depending on temperature. Since the amount of ammonium sulfate, ammonium bisulfate, and ammonium bisulfite is proportional to the amount of SO3 present, more deposits can theoretically form as a result of this location. However these deposits will form in the colder regions downstream of the CO and SCR catalysts, and therefore do not affect the performance of either catalyst.
Placing the CO catalyst in a low-temperature region may also result in other problems–such as plugging of the CO catalyst with ammonium salts. To the best of my knowledge, only one manufacturer is experimenting with a CO catalyst in a temperature range less than 400F, but the ammonia slip and sulfur must be negligible.
The decision as to where to locate the CO catalyst should be based on assessing all the risks, costs, and effects on performance. The analysis should also take into consideration all parameters–including catalyst volume, exhaust-gas pressure drop, deposit formation, current technology, OEM warranty, and reliability.
Repair of a fertiliser prill tower
Fertiliser is often sold as small pellets known as ‘prills’, formed by pumping hot liquid fertiliser through spray heads at the top of a tall tower. As the fertiliser falls down the drop shaft, it cools and solidifies to form prills, which are directed by conveyor from a collection hopper at the tower base to the packing and despatch area. Fertilisers are frequently based on ammonium nitrate or urea. The same prilling technique can be used for either of these processes.
While urea and ammonium nitrate are valuable commodities as fertilisers, they present an aggressive environment to concrete. Protective design measures are required, together with ongoing maintenance to repair the affected areas.
One of the earliest operating ammonium nitrate prill towers still in existence is operated by Terra Nitrogen (UK) Ltd at Severnside, Hallen, near Bristol. Constructed in 1964, it consists of a 15.6m-diameter reinforced concrete cylinder, 178mm thick and 86m high, on concrete piled foundations. A five-storey reinforced concrete plant house, supported on the drop shaft, lift shaft and a single column, forms the upper section of the tower from 75m to 100m (see Figure 1).
Ammonium nitrate attack
The primary deterioration mechanism is the reaction of ammonium nitrate with the calcium hydroxide in the cement paste to form calcium nitrate. This leads to a reduction in the calcium hydroxide content, together with an associated increase in porosity and decrease in alkalinity. The calcium nitrate then reacts with hydrated calcium aluminate, present in cement, to form calcium nitroaluminate. The resultant volumetric increase associated with this reaction leads to an expansion of the cement matrix and subsequent bursting of contaminated layers.
Ammonium nitrate attack is generally regarded as a removal of surface laitance followed by a loss of aggregate from the weak cement matrix. In time, the concrete can be attacked throughout its full thickness. Where alternating wetting and drying conditions exist, the surface laitance may not be removed, leading to the bursting of an apparently sound concrete surface. This may be confused with the more familiar cracking and spalling that arise from reinforcement corrosion.
As little as 0.5% by weight of concrete can cause significant damage over time, and the degradation increases with ammonium nitrate concentration. Of more concern in ammonium nitrate environments is the potential for stress corrosion of the steel reinforcement. This is coupled with tensile stress, which causes brittleness and results in direct tensile failure of reinforcement. The likelihood of stress corrosion increases if the steel has surface defects. Only a short initial crack length is required for stress-corrosion cracking to propagate. Factors affecting the rate of deterioration include temperature, residual surface stresses, surface defects, roughness and topography, chemical composition, diameter and ultimate tensile strength. Stress cracking at Severnside was particularly evident in links that, when exposed to ammonium nitrate attack, were frequently found to have broken at positions where they had been bent and subjected to high residual stresses. The combination of concrete softening and stress causing the corroded steel to crack presents a significant structural concern because of the reduction in ductility of reinforced concrete sections and their ability to redistribute loads.
Repair history
Ammonium nitrate attack has been known in the fertiliser industry for some years, and protective measures were taken during the original design of the Severnside tower. The general approach to concrete repairs has been to remove degraded concrete and replace it with either epoxy or polymer-modified concrete. Such repairs tend to fail at the interface with the original concrete owing to the difficulty of removing all the contamination. Subsequent repair requires the removal of further degraded concrete until eventually it becomes necessary to carry out a full structural repair or element replacement. Examples of previous major repairs have included the reconstruction of the concrete roofs at the 86m and 100m levels and the spraying on of a reinforced silica fume concrete jacket to the outside of the drop shaft over its full height.
Plant house integrity project
Continued confidence in the nitrate market led Terra to specify an extended service life of 15 years from the tower. Their technical advisors, ABB Eutech, identified that major work was necessary and produced a repair strategy to avoid loss of production. Taylor Woodrow’s engineering and consultancy team were subsequently appointed to undertake the project management, from detailed investigation, testing and assessment, through design to supervision of remedial works.
The project team - comprising Terra, ABB Eutech, Taylor Woodrow and main contractors Bierrum - were able to draw on the combined strength and experience of all parties involved in tackling this project.
Sensitivity of Ziehl-Neelsen method for centrifuged deposit smears of sputum samples transported in cetyl-pyridinium chloride
Background & objectives: Sensitivity of Ziehl-Neelsen (ZN) method is known to be low for liquefied sputum smears. Information on the ZN sensitivity for centrifuged deposit smears is not known. This study was carried out to determine the sensitivity of ZN method for acid fast bacilli (AFB) in centrifuged deposit smears and liquefied sputum smears made from sputum samples transported in cetyl-pyridinium chloride (CPC) solution.
Methods: Liquefied sputum smears and the corresponding centrifuged deposit smears from each of the 607 consecutive sputum samples collected from tuberculosis patients admitted to receive treatment transported in CPC were read by the same readers and their results compared with culture results.
nterpretation & conclusion: Our study demonstrated that the sensitivity of ZN method for AFB in centrifuged deposit smears and liquefied sputum smears was reduced if sputum samples are transported in CPC solution.
VEB-1 in Achromobacter xylosoxidans from cystic fibrosis patient, France
Multidrug-resistant Achromobacter xylosoxidans was recovered from the sputum of a patient with cystic fibrosis. The VEB-1 extended-spectrum [beta]-lactamase was detected on a class 1 integron. This first report of a VEB-1–producing isolate in this population requires further investigation to determine its distribution.
Achromobacter (formerly Alcaligenes) xylosoxidans is a newly emerging microorganism isolated with increased frequency from the lungs of patients with cystic fibrosis (CF), but information about its clinical relevance is limited (1). A. xylosoxidans is innately resistant to many antimicrobial drugs (2), except piperacillin, piperacillin-tazobactam, and imipenem, and moderately susceptible to ceftazidime (45% of susceptible isolates), which is widely used to treat infection due to Pseudomonas aeruginosa (3,4). The mechanisms involved in cases of high-level resistance to ceftazidime have not been described for A. xylosoxidans. Possible mechanisms for ceftazidime resistance among gram-negative bacilli are alterations in outer membrane proteins, overproduction of cephalosporinase, or production of an extended-spectrum [beta]-lactamase (ESBL). ESBLs are enzymes distributed worldwide (5) that hydrolyze oxyimino-cephalosporins and monobactams and are susceptible to [beta]-lactamase inhibitors such as clavulanic acid and tazobactam. We report on the isolation from a CF patient of A. xylosoxidans that produced the VEB-1 ESBL. This is the first report of ESBL production in A. xylosoxidans and the first report of a VEB-1–producing isolate from a CF patient.
During the past 10 years in our 1,600-bed university hospital, 37 CF patients had [greater than or equal to] 1 respiratory tract specimen that contained A. xylosoxidans. Preliminary pulsed-field gel electrophoresis of these strains has failed to identify shared isolates among the patients, but studies are ongoing. In November 2003, A. xylosoxidans 476 (AX476) was isolated from the sputum of a 17-year-old male CF patient. This patient had good pulmonary function (forced expiratory volume 1 = 99% of predicted value), had never been colonized or infected by P. aeruginosa, and therefore never received ceftazidime. The strain was identified with the Api 20NE system (bio-Merieux, Marcy-l’Etoile, France), and antimicrobial susceptibility testing was performed and interpreted as recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) (6).
The antibiogram, which was performed by a disk diffusion method, showed AX476 to be highly resistant to ceftazidime, aminoglycosides, sulfonamides, trimethoprim, and ciprofloxacin but fully susceptible to tetracyclines, piperacillin/tazobactam, ticarcillin/clavulanic acid, and imipenem. Because of an unusual synergy between ticarcillin and ticarcillin/clavulanic acid (Figure), we compared the inhibition zones of third-generation cephalosporin disks with and without clavulanic acid (BioRad, Marnesla-Coquette, France). The zones were 7 mm for ceftazidime and 19 mm for ceftazidime plus clavulanic acid (Figure), which strongly indicated production of an ESBL. Isoelectric focusing showed that AX476 produced a [beta]-lactamase with an isoelectric point of 7.4. A large plasmid of [approximately equal to] 200 kb (pJDB 1) was easily transferred by conjugation to Escherichia coli K-12 C600. The transconjugants, E. coli (pJDB1) sorbitol-fermenting, which were selected on MacConkey agar that contained 4 [micro]g/mL of ceftazidime, were resistant to sulfonamides and trimethoprim, had reduced susceptibility to aminoglycosides, and harbored a [beta]-lactamase with an isoelectric point of 7.4. The resistance phenotype of the isolate and the value of the isoelectric point of the enzyme suggested the production of the ESBL VEB-1 (7).
The MICs for [beta]-lactams for AX476 and its transconjugant, determined by Mueller-Hinton broth dilution method, are shown in Table 1. By using [blav.sub.VEB-1]-specific primers, a positive PCR result was obtained on total DNA from AX476 and the transconjugants. All genetic analyses of [bla.sub.VEB-1] published so far have identified either its chromosome (8) or its plasmid (9) location and mostly its integration within class 1 integrons of variable structure. Integrons are potentially mobile genetic elements that comprise conserved sequences that flank a variable region and may contain inserted antimicrobial drug resistance gene cassettes (10). The 5′-conserved segment includes the gene intI1 that encodes an integrase, the cassette integration site attI1, and a promoter responsible for the expression of the genes located downstream within the variable region. The 3′-conserved region contains either a qacE[DELTA]1 gene that encodes resistance to quaternary ammonium compounds or a combination of 3 genes: qacE[DELTA]1, sulI (which encodes resistance to sulfonamides), and orf5 (an open reading frame of unknown function).
To search for the presence of such a class 1 integron in AX476 and its transconjugant, we performed PCR on total DNA of AX476 and E. coli (pJDB 1) by using the primers L1 and R1 specific for the detection of class 1 integrons (11). We obtained a fragment of 2.3 kb in the clinical strain and its transconjugant, which was sequenced on both strands. By using a set of primers, we deduced the structure of this integron (Table 2). Three gene cassettes have been identified. The first, dhfr (dihydrofolate reductase), encoded a putative trimethoprim-resistance protein. This dhfr was identical to that reported in Salmonella enterica serovar Typhi (GenBank accession no. AL513383) and to the dhfr gene cassette contained in a class 1 integron from Klebsiella pneumoniae not yet published (GenBank accession no. AJ971342). The second cassette, [blav.sub.VEB-1], encoded the ESBL VEB-1 first described in E. coli (7). The third and last gene cassette was aadB. It encoded an aminoglycoside adenyltransferase that conferred resistance to kanamycin, gentamicin, and tobramycin and was identical to other sequenced aadB gene cassettes located on integrons containing [bla.sub.VEB-1] gene (7,8). VEB-1 has been detected in Enterobacteriaceae and P. aeruginosa isolates from Southeast Asia (9) but never in A. xylosoxidans. In France, VEB-1-producing isolates of Acinetobacter baumannii have been involved in several outbreaks of nosocomial infection in intensive care units (12,13); however, we have not yet detected a VEB-1–producing isolate in our hospital.
Combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines
Recombinant, immunodominant antigens derived from Mycobacterium tuberculosis can be used to effectively vaccinate against subsequent infection. However, the efficacy of these recombinant proteins is dependent on the adjuvant used for their delivery. This problem affects many potential vaccines, not just those for tuberculosis, so the discovery of adjuvants that can promote the development of cell-mediated immunity is of great interest. We have previously shown that the combination of the cation ic surfactant dimethyl dioctadecyl ammonium bromide and the immunomodulator modified lipid A synergistically potentiates ThI T-cell responses. Here we report a screening program for other adjuvants with reported Th1-promoting activity and identify a second novel adjuvant formulation that drives the development of ThI responses with an extremely high efficacy. The combination of dimethyl dioctadecyl ammonium bromide and the synthetic cord factor trehalose dibehenate promotes strong protective immune responses, without overt toxicity, against M. tuberculosis infection in a vaccination model and thus appears to be a very promising candidate for the development of human vaccines.-Authors’ Abstract.
perchlorate pollutant masculinizes fish
Known largely as a component of rocket fuel, perchlorate is a pollutant that often turns up in soil and water. In dozens of studies, it has perturbed thyroid-hormone concentrations, which can affect growth and neurological development. Data from fish now indicate that perchlorate can also disrupt sexual development.
Some of the changes were so dramatic that scientists initially mistook female fish for males. Several females displayed male-courtship behavior and produced sperm.
Richard R. Bernhardt of the University of Alaska in Anchorage and his colleagues focused on threespine sticklebacks (Gasterosteus aculeatus), a tiny marine species. For 3 weeks, the researchers incubated wild-captured adults in clean water or in water treated with 30, 60, or 100 parts per million (ppm) perchlorate. The adults spawned during that period.
Each group’s offspring were then raised to sexual maturity in similarly treated or untreated water. At spawning age, 10 apparent males per treatment group were each given their own aquariums. Once a day, each male received a 10-minute visit from an egg-swollen female in the same treatment group.
The first sign of something amiss: Among perchlorate-exposed fish, many would-be dads lacked the electric-blue and red coloration that normally signals readiness to spawn. Most of these fish didn’t exhibit typical reproductive behaviors, such as nest building, and many ignored prospective mates. Among cleanwater males, 80 percent spawned. As the perchlorate concentration climbed from 30 to 100 ppm, successful spawning fell from 50 percent to zero.
Eventually, the bellies of three apparent males began swelling with eggs. They proved to be hermaphroditic females, bearing both fertile eggs and fertile sperm.
The perchlorate-exposed true males developed unusually long testes.
Last January, the Environmental Protection Agency suggested limiting perchlorate contamination in natural bodies of water to 24.5 parts per billion. The concentrations used in the new study were at least 1,000 times that limit.
However, these doses are still environmentally relevant, argue aquatic toxicologist Bernhardt and his colleagues in the August Environmental Toxicology and Chemistry. They say that the test concentrations are similar to or less than those at several contaminated U.S. sites.
The “big surprise” was that perchlorate could produce hermaphroditic females and males with superlarge testes, says ecotoxicologist Gerald T. Ankley of EPA’s Mid-Continent Ecology Division in Duluth, Minn. Clearly, that’s “not something you would have anticipated [from] the way we think perchlorate works,” he adds.
All the changes observed suggest that perchlorate “is acting like an androgen,” or male-sex hormone, notes fish physiologist Ann Cheek of the University of Texas Health Science Center at Houston. Confirming this would require cellular analyses of testes and thyroid tissue.
Christopher W. Theodorakis of Southern Illinois University in Edwardsville argues that the “intriguing” masculinization may instead point to a new role for thyroid hormones–preservation of reproductive function.
“This paper may be telling us there’s more to perchlorate–and its effects on the thyroid–than we’d realized,” agrees R. Thomas Zoeller, a thyroid endocrinologist at the University of Massachusetts in Amherst. That “could be pretty profound,” he says.
Effects of ammonium perchlorate on thyroid function in developing fathead minnows, pimephales promelas
Perchlorate is a known environmental contaminant, largely due to widespread military use as a propellant. Perchlorate acts pharmacologically as a competitive inhibitor of thyroidal iodide uptake in mammals, but the impacts of perchlorate contamination in aquatic ecosystems and, in particular, the effects on fish are unclear. Our studies aimed to investigate the effects of concentrations of ammonium perchlorate that can occur in the environment (1, 10, and 100 mg/L) on the development of fathead minnows, Pimephales promelas. For these studies, exposures started with embryos of < 24-hr postfertilization and were terminated after 28 days. Serial sectioning of thyroid follicles showed thyroid hyperplasia with increased follicular epithelial cell height and reduced colloid in all groups of fish that had been exposed to perchlorate for 28 days, compared with control fish. Whole-body thyroxine ([T.sub.4]) content (a measure of total circulating [T.sub.4]) in fish exposed to 100 mg/L perchlorate was elevated compared with the [T.sub.4] content of control fish, but 3,5,3′-triiodothyronine ([T.sub.3]) content was not significantly affected in any exposure group. Despite the apparent regulation of [T.sub.3], after 28 days of exposure to ammonium perchlorate, fish exposed to the two higher levels (10 and 100 mg/L) were developmentally retarded, with a lack of scales and poor pigmentation, and significantly lower wet weight and standard length than were control fish. Our study indicates that environmental levels of ammonium perchlorate affect thyroid function in fish and that in the early life stages these effects may be associated with developmental retardation. Key words: development, endocrine disruption, fathead minnow, perchlorate, thyroid, thyroxine, triiodothyronine.
In recent years there has been increasing concern about the presence of perchlorate in ground and surface waters and the percolation of perchlorate into drinking waters [Urbansky 1998; U.S. Environmental Protection Agency The major source of ground and surface water contamination is ammonium perchlorate, the primary ingredient of the solid propellant in rockets and missiles (Logan 2001; U.S. EPA 2002). Perchlorate salts are also used in smaller amounts as components of air bag inflators, road flares, and fireworks; in electroplating and in tanning and finishing leathers; and as mordants for fabrics and in producing paints and enamels (Logan 2001; U.S. EPA 2002). Discharge from rocket fuel manufacturing plants, demilitarization of weapons, and the washing out and refueling of rockets are responsible for most of the ammonium perchlorate released into the environment (Urbansky 1998; U.S. EPA 2002). Indeed, at the Longhorn Army Ammunition Plant in Texas (USA), perchlorate has been measured at 30-31 mg/L in a water treatment holding pond (Smith et al. 2001).
Perchlorate has several chemical properties that make environmental contamination difficult to resolve and decontamination difficult to achieve (Logan 2001). The perchlorate anion is persistent because of its tetrahedral structure (Wolff 1998). Perchlorate salts completely ionize in solution, and the perchlorate anion is highly mobile (Logan 2001). As a result of these properties, groundwater contamination inevitably presents a risk to drinking water quality, and perchlorate has been detected in many drinking water supplies. In Nevada, 4-24 [micro]g/L was detected in drinking water (Xiao et al. 2001), and in California a number of drinking water wells showed peaks of 4-820 [micro]g/L (California Department of Health Services 2004). As a result, the U.S. EPA has estimated that perchlorate affects the quality of drinking water for 15 million people in the United States (Logan 2001).
Based on U.S. EPA guidance, and assessment of toxicity data, several U.S. states have set advisory levels for perchlorate in drinking water that vary between 1 and 18 [micro]g/L. The most recent reappraisal in California set a public heath goal for drinking water (maximum contaminant level) of 6 [micro]g/L (Office of Environmental Health Hazard Assessment 2004).
There is a long history of clinical use of perchlorate as a pharmacologic inhibitor of thyroid hormone synthesis (Hobson 1961; Wolff 1998). Thyroid gland follicles trap iodide required for the iodination of tyrosine molecules. The resulting iodothyronines are then reversibly combined with the storage protein, thyroglobulin, within the lumen of each of the thyroid follicles (Leatherland 1988, 1993). Perchlorate competitively inhibits iodide uptake by the sodium/iodide symporter at the basolateral membrane of the follicles (Capen 1997; Wolff 1998) and induces iodide efflux from the follicles by an as yet unexplained mechanism (Wolff 1998). These pharmacologic actions might be predicted to reduce circulating levels of thyroid hormones, and several studies in mammals given drinking water containing perchlorate at target doses of 0.01-100 mg/kg/day .
Histology. Whole fish (n = 5) fixed in formalin were decalcified for 14 days in 5% formic acid in 5% formaldehyde. Fish were wax embedded and serially sectioned (6/am) through all the thyroid follicles. Each follicle in each fish (5-13 follicles/fish) was traced through its entirety, and epithelial cell height was measured at the largest point.
Thyroid hormone extractions. Thyroid hormones were extracted from fathead minnow larvae based on the technique described by Greenblatt et al. (1989). Larvae were placed in Teflon tubes on ice, and 2 mL 95% ethanol containing 1 mM 6-N-propyl-2-thiouracil (PTU) was added. Samples were homogenized (Ultra Turax T25; Janke and Kunkel, Staufen, Germany) and sonicated for 20 sec (Vibra-Cell, 50% output; Sonics and Materials, Meryin/Satigny, Switzerland). A further 2 mL of 95% ethanol with 1 mM PTU was added, and samples were vortexed. Samples were centrifuged for 10 min (10,000g; 4[degrees]C), the supernatant was decanted into clean Teflon tubes, and 2 mL 95% ethanol containing PTU was added to the pellets. Tubes were vortexed vigorously and recentrifuged for 10 min (10,000g, 4[degrees]C). Supematants were pooled and evaporated to dryness under nitrogen, and desiccated samples were resuspended in 0.25 mL barbital buffer containing 2.5 mg/mL anilino naphthalene sulfonic acid (to disrupt the coupling between thyroid hormones and serum proteins, including lipoproteins), 0.25 mL ethanol, and 1 mL chloroform. Tubes were vigorously vortexed and then centrifuged for 10 min (1,500g; 4[degrees]C), producing two phases. The top ethanolic layer was removed using a glass pipette for radioimmunoassay (RIA) of thyroid hormones. The recovery of thyroid hormones was determined by addition of radioiodinated [T.sub.4] or [T.sub.3] after homogenization of whole larvae (n = 5). The recovery of 59.5 [+ or -] 3.25% [T.sub.4] and 63.9 [+ or -] 3.27% [T.sub.3] was comparable with those recoveries reported for larvae of other fish species.
Extracted samples or standard solutions (30 [micro]L) were incubated at 4[degrees]C overnight (in triplicate) with 100 [micro]L antiserum and 100 [micro]L radioiodinated solution, with additional “total counts” and “blank” tubes. The next morning, free and bound hormones were separated by addition of 100 [micro]L Sac-Cel (Immunodiagnostic Systems Limited, Tyne and Wear, UK) and a solution of cellulose-coupled antibodies (anti-sheep/goat); tubes were centrifuged, and the pellet of bound radiolabeled hormone was counted (Cobra gamma counter; Packard, Boston, MA, USA).
Ammonium nitrate a possible source - Carcinogens
Farmers use ammonium nitrate as a crop fertilizer and plants produce it to use as an energy source in the absence of light. The problem is that nitrate could be responsible for starting a chain of events in the body that leads to the formation of cancer-causing agents, according to Richard Loeppky, professor of chemistry, University of Missouri-Columbia. When nitrate is ingested, either through eating or drinking contaminated water, it is transferred into the bloodstream through normal digestive processes. Some of the nitrate then is transported to the salivary glands where bacteria transform it into nitrite. The nitrite is swallowed into the stomach again where it bonds with amino acids that are not combined with any particular protein. At this point, nitrolic acid is formed. This newly identified compound has properties that could lead to the formation of cancer in various areas of the body.
“The stomach is an ideal reactor for the kinds of chemistry that we are talking about,” Loeppky explains. “In addition, other researchers have suspected that amino acids played a role in forming these cancerous compounds, but until we made this discovery, no one had much of an idea how this happened.”
Nitrate also might combine with other compounds in the stomach to form cancer-causing agents, many of which must be metabolized before they are able to induce any damage. Because of this, Loeppky recommends that producers and consumers limit their use of nitrates as much as possible. “While we don’t know much about this newly discovered compound, the evidence suggests that it can alter or damage DNA. What is completely unknown is the pathway on how this is accomplished”.