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Environmental hazard of yperite released at sea: Sublethal toxic effects on fish
Abstract
The aim of this study was to evaluate the potential toxicological effects on fish related to the leakage of yperite from rusted bomb shells dumped at sea. Both in vivo and field studies have been performed. As for the in vivo experiment, specimen of European eel were subcutaneously injected with 0.015, 0.15 and 1.5mg/kg of yperite and sacrificed after 24 and 48h. In the field study, specimen of Conger eel were collected from a dumping site in the Southern Adriatic Sea. The presence/absence of yperite in tissues, genotoxicity, detoxification enzymes, histological alterations and gross abnormalities were investigated. Results of the in vivo experiment showed a significant increase of EROD activity at both 24h and 48h. UGT activity increased significantly at 48h post injection. An acute inflammatory response after 24h in skin layers and muscle was observed, associated to cell degeneration and necrosis after 48h at the highest dose. On field, comet assay revealed genotoxicity in gills of fish from the dumping site. Specimen from the dumping site showed significantly higher EROD activities compared to controls, deep ulcers and papules on skin together with liver and spleen histopathological lesions.
... The comet assay results revealed that DNA strand breaks were significantly higher in gill cells of fish from dumping area when compared with the reference site. On the contrary, comet assay results did not reveal any significant difference between liver and kidney cells of fish from the dumping and control site (Della Torre et al., 2013). ...
... First, MN induction is a cell cycle-dependent phenomenon and low frequency of micronuclei and other nuclear lesions would require the use of impractically large sample sizes to evaluate the marginally contaminated sites. DNA repair mechanisms or fast removal of apoptotic cells might also reduce the presence of micronuclei in the tissue samples, leading to underestimation of actual genotoxic impact (Belpaeme et al., 1996;Della Torre et al., 2013). In this work, there was a strict limitation for sample size because of DOE's species protection legislations. ...
... Thus, the possible reason for the observed discrepancy between the two endpoints could be the higher vulnerability of gill cells as compared to blood and liver. Gills are the first target of waterborne toxics, because they are the primary site of uptake of substances dissolved in water and have vast surface areas in direct contact with genotoxicants (Della Torre et al., 2013). The kidney is an inappropriate model tissue for measurement of genotoxicity in this study. ...
... Della Torre et al. (2010Torre et al. ( , 2013 have identified DNA damage (Comet assay, quantified as 28.77% and 28.51% DNA migrated in the tail) in gills of conger fished at a CW dumping site in the Mediterranean Sea and Southern Adriatic Sea. ...
... The highest ∑Gtox and ∑Cytox levels were recorded at 10 sampling stations close to CW and at 19 stations close to conventional munitions dumpsites: for herring at nine stations (3a, 4a, 1f, 4s, 1w, 1B, 6F, 7K and 41P), for flounderat six stations (3a, 4a, 9K, 1M and 41P) and for cod at two stations (6F and 7K). Della Torre et al. (2010Torre et al. ( , 2013 investigated genotoxicity effects in European conger (Conger conger) gills, which is one of the target tissues of CWAs. The study reported deep dermal, external and internal organ damage in liver and spleen and significantly high EROD activity in blackbelly rosefish (Helicolenus dactylopterus) and European conger, caught near CW dumping site in the Southern Adriatic Sea. ...
Frequencies of eight nuclear abnormalities (NAs) reflecting environmental genotoxicity and cytotoxicity, were examined in 739 specimens of herring (Clupea harengus), flounder (Platichthys flesus) and cod (Gadus morhua) collected between 2009 and 2017 at 50 study stations located in the Gulf of Gdansk and the southern part of the Gdansk Deep (the Baltic Sea). The highest levels of geno-cytotoxicity were recorded in fish caught at stations located in close proximity to chemical and conventional munitions dumping sites or in zones polluted by Chemical Warfare Agents (CWAs). The values of NAs were significantly higher (up to 51-fold compared with the reference level) in herring caught at seven stations and in cod from one station located close to the munitions dumping sites. Exceptionally high total genotoxicity (∑Gtox) risk was found for flounder collected from 18 (72.0%) stations, herring caught at 12 (38.7%) stations and cod caught at four (17.4%) studied stations.
... Given the dilutional power of the massive volume of ocean water, agents that undergo rapid dissolution and hydrolysis should result in less long-term toxicity than those agents that are environmentally persistent. [33,34] Phosgene, chloroacetophone, and blood agents have hydrolysis and dissolution rates that render them unlikely to chronically threaten underwater ecosystems. LD50 or LCt50 values may be unknown or may be derived from animal models only. ...
... It is a lipophilic substance with low volatility and weak aqueous solubility. [34] The chemical's low solubility in water enhances the potential for environmental persistence. [36] Sulfur mustard dissolution is slow, but once dissolved hydrolyses rapidly to form thiodiglycol and thioxane in a 4:1 ratio, with the formation of intermediate sulfur compounds in smaller amounts. ...
Introduction:
Chemical weapons dumped into the ocean for disposal in the twentieth century pose a continuing environmental and human health risk.
Objective:
In this review we discuss locations, quantity, and types of sea-dumped chemical weapons, related environmental concerns, and human encounters with sea-dumped chemical weapons.
Methods:
We utilized the Ovid ( http://ovidsp.tx.ovid.com ) and PubMed ( http://www.pubmed.org ) search engines to perform MEDLINE searches for the terms 'sea-dumped chemical weapons', 'chemical warfare agents', and 'chemical munitions'. The searches returned 5863 articles. Irrelevant and non-English articles were excluded. A review of the references for these articles yielded additional relevant sources, with a total of 64 peer-reviewed articles cited in this paper.History and geography of chemical weapons dumping at sea: Hundreds of thousands of tons of chemical munitions were disposed off at sea following World War II. European, Russian, Japanese, and United States coasts are the areas most affected worldwide. Several areas in the Baltic and North Seas suffered concentrated large levels of dumping, and these appear to be the world's most studied chemical warfare agent marine dumping areas. Chemical warfare agents: Sulfur mustard, Lewisite, and the nerve agents appear to be the chemical warfare agents most frequently disposed off at sea. Multiple other type of agents including organoarsenicals, blood agents, choking agents, and lacrimators were dumped at sea, although in lesser volumes. Environmental concerns: Numerous geohydrologic variables contribute to the rate of release of chemical agents from their original casings, leading to difficult and inexact modeling of risk of release into seawater. Sulfur mustard and the organoarsenicals are the most environmentally persistent dumped chemical agents. Sulfur mustard in particular has a propensity to form a solid or semi-solid lump with a polymer coating of breakdown products, and can persist in this state on the ocean floor for decades. Rates of solubility and hydrolysis and levels of innate toxicity of a chemical agent are used to predict the risk to the marine environments. The organoarsenicals eventually breakdown into arsenic, and thus present an indefinite timeline for contamination. Generally, studies assaying sediment and water levels of parent chemical agents and breakdown products at dumpsites have found minimal amounts of relevant chemicals, although arsenic levels are typically higher in dumpsites than reference areas. Studies of marine organisms have not shown concerning amounts of chemical agents or breakdown products in tissue, but have shown evidence of chronic toxicity. There is believed to be minimal risk posed by seafood consumption. Microbiota assays of dumpsites are significantly altered in species composition compared to reference sites, which may imply unseen but significant changes to ecosystems of dumpsites. Human health concerns: The major human health risk at this time appears to arise from acute exposure to an agent by either accidental recovery of a chemical weapon on a fishing vessel, or by munitions washed ashore onto beaches.
Conclusions:
Improving technology continues to make the deep sea more accessible, thus increasing the risk of disturbing munitions lying on or buried in the seabed. Pipe laying, cable burying, drilling, scuba diving, trawling, and undersea scientific research are the activities posing the most risk. The long-term threat to the benthic habitat via increased arsenic concentrations, shifts in microbiota speciation, and chronic toxicity to vertebrates and invertebrates is not currently understood. The risk to the environment of massive release via disturbance remains a distinct possibility. Terrorist recovery and re-weaponization of chemical agents is a remote possibility.
... Attributed to the leakage of yperite from rusted bomb shells dumped in the Southern Adriatic Sea, 3e4 times higher levels of As and Hg were recorded in fillets of two benthic fish species and genotoxicity effects of CWA were described. The suitability of fish for the environmental quality assessment was emphasised (Della Torre et al., 2010 Torre et al., , 2013). The main objective of the present study was to evaluate the level of environmental genotoxicity and cytotoxicity in three native fish species inhabiting the chemical munitions dumping zones located in the Bornholm Basin of the Baltic Sea. ...
... DNA damage (quantified as 28.77% and 28.51% DNA migrated in the tail) was found only in gills of conger fished at CW dumping site (Della Torre et al., 2010 Torre et al., , 2013). Experimental treatment with different concentrations of NaAsO 2 evoked elevated micronuclei induction in blood erythrocytes of fish Oreochromis mossambicus after 96 and 192 h. ...
The data on environmental genotoxicity and cytotoxicity levels as well as on genotoxicity risk in flounder (Platichthys flesus), herring (Clupea harengus) and cod (Gadus morhua) collected in 2010-2012 at 42 stations located in chemical munitions dumping areas of the southern Baltic Sea are presented. The frequency of micronuclei, nuclear buds and nucleoplasmic bridges in erythrocytes was used as genotoxicity endpoint and the induction of fragmented-apoptotic, bi-nucleated and 8-shaped erythrocytes as cytotoxicity endpoint. The most significantly increased geno-cytotoxicity levels were determined in fish collected near known chemical munitions dumpsites. Extremely high genotoxicity risk for flounder were identified at 21 out of 24 stations, for herring at 29 out of 31 and for cod at 5 out of 10 stations studied. The reference level of genotoxicity was not recorded at any of the stations revealing that in the sampling area fish were affected generally.
... [52], VX demonstrates an uncommon characteristic of increased water solubility at lower temperatures. [53], with its slow hydrolysis rate, VX is anticipated to have a subsea half-life of 5.4 years. The initial hydrolysis products retain toxicity and acetylcholinesterase activity, thereby prolonging potential adverse effects on marine ecosystems. ...
Quantitative Structure-Activity Relationship (QSAR) models are essential in predicting the characteristics of chemical warfare agents (CWAs), offering crucial insights into their biological, biochemical, and environmental activities. This paper examines how QSAR models elucidate the complex relationships between molecular structures and CWA actions. By leveraging principles from biology, biochemistry, and environmental research, QSAR models accurately predict key features such as CWA toxicity, reactivity, and environmental persistence. This study explores the fundamental mechanisms behind CWA interactions with biological systems, molecular targets, and environmental compartments, highlighting the potential of QSAR models to guide the development of novel antidotes, decontamination strategies, and environmental monitoring protocols. Integrating insights from various disciplines, this work underscores the significance of QSAR modeling in enhancing our understanding of CWA properties and supporting informed decision-making in defense, public health, and environmental management.
... En effet, environ 50000 tonnes d'armes chimiques (armes, munitions, équipements associés vides ou chargés) et environ 15000 tonnes d'agents chimiques ont été déversées dans ces zones [22]. La problématique de la contamination de la mer, des organismes marins et même de l'homme suite à la consommation du poisson péché dans la région a été traitée [23,24,25,26,27]. C'est dans ce cadre que plusieurs projets de recherche ont été lancés comme le projet «CHEMSEA» (Chemical Munitions Search & Assessment) financé par l'Union Européenne qui porte sur l'amélioration des méthodes d'analyse et de détection de ces composés et sur l'évaluation de leur impact possible sur l'environnement [28]. ...
Les agents neurotoxiques organophosphorés (OPNA) sont les agents de guerre les plus toxiques. Ils se dégradent rapidement dans la nature ou dans le corps humain par hydrolyse pour donner naissance à des acides alkyl méthyl phosphoniques (AMPA). Ces produits de dégradations peuvent être utilisés comme marqueurs d’une exposition aux OPNA. Cependant, leur analyse par chromatographie liquide couplée à la spectrométrie de masse (LC-MS) dans des matrices complexes implique le recours à des méthodes de traitement de l’échantillon efficaces. Ainsi, l'extraction liquide assistée par membrane parallèle artificielle (PALME) a été développée pour extraire et concentrer les AMPA. Il s’agit d’une méthode de microextraction réalisée dans un système de deux plaques 96-puits où l'échantillon aqueux et la phase acceptrice aqueuse sont séparés par une membrane plate imprégnée de solvant organique. Ce qui permet le traitement de plusieurs échantillons simultanément. Initialement, un système de plaques tel que décrit dans la littérature a été utilisé. Plusieurs paramètres affectant l’extraction des analytes ciblés ont été étudiés dont, pour la première fois, de nouveaux tel que l’effet de la force ionique et la température sur l’extraction. Cependant, les facteurs d’enrichissements (FE) obtenus étaient limités et ne permettaient pas d’atteindre la sensibilité souhaitée et ce malgré les taux d’extraction exhaustifs pour 3 des 5 composés étudiés. Suite à ce constat, le dispositif de la PALME a été modifié en introduisant une plaque 96-puits donneuse avec un volume donneur 6 fois plus important que la plaque 96-puits classique de 0.5 ml de volume. Ceci a permis d’atteindre des FE de 42. La méthode PALME finale développée a donc pu être appliquée pour la première fois au domaine environnemental sur des échantillons d’eau de rivière, d’eau usée et d’extraits aqueux de sols.Afin de répondre aux 10 principes de traitement de l’échantillon vert, une partie de ces travaux de thèse a eu pour objectif de rendre la PALME plus « verte » grâce au remplacement des solvants organiques, nuisibles à la santé et l’environnement, par des solvants naturels. L’octanol utilisé comme solvant en PALME a donc été remplacé par un solvant eutectique profond hydrophobe d’origine naturelle (NaHDES) à base de thymol et de coumarin. Cette nouvelle méthode NaHDES-PALME associée à la LC-MS/MS a été appliquée avec succès sur des échantillons biologiques (urines). Elle présente une sensibilité conforme aux exigences de l'OIAC et peut donc être utilisée à des fins médicales et/ou criminalistiques. Elle est très simple à mettre en œuvre, économique, rapide, verte et à haut débit et donc totalement adaptée au contexte d’urgence d’une intervention sur une scène potentiellement contaminée par les OPNA. Par ailleurs, les paramètres opérationnels considérés comme optimaux pour l’extraction des AMPA en PALME se sont avérés être les mêmes que ceux préalablement obtenus lors de l’optimisation de la méthode de micro-extraction liquide assistée par fibre creuse (HF-LPME), méthode similaire dans son principe mais qui diffèrent en termes de dimensions. Ce résultat permet d’envisager le transfert de très nombreuses méthodes déjà développées en HF-LPME vers la PALME.
... Evidence for this assumption were shown by studies indicating a possible impact on the marine biota near dumpsites. For example, different species of fish (Gadus morhua, Clupea harengus, Platichthys flesus) caught in the southern part of the Baltic Sea revealed high geno-and cytotoxicity levels, while blackbelly rosefish (Helicolenus dactylopterus) and european conger (Conger conger) caught in the Mediterranean Sea showed dermal blistering, as well as DNA-and histopathological damage (Bar� sien _ e et al., Della Torre et al., 2013. For the Skagerrak, the transition zone between North and Baltic Sea, Tørnes et al. (2002) reported that the fauna investigated around shipwrecks at the CWA dumping ground in the Skagerrak seemed to be generally healthy. ...
The seas worldwide are threatened by a “new” source of pollution. Munitions dumped into the seas worldwide will corrode and start to leak. Their impacts on the environment and on human health are now more than ever subject of scientific research. Bivalves are a first choice bioindicator and their importance is demonstrated in numerous worldwide studies as well as their integration in important monitoring programs. In this review, the use of mussels in context with marine pollutants in recent years is pointed out in general but with a special focus on dumped conventional and chemical munitions. Monitoring experiments with mussels are able to generate large data sets, which should be mandatory included in decision support tools to increase their weight of evidence. The usefulness of mussels with regard to dumped munitions has clearly been documented in recent years and the further application of this important biomonitoring system is strongly recommended.
... The environmental toxicity of Chemical warfare agents (CWAs) has in recent years received more attention (Della Torre et al. 2013;Sanderson et al. 2014) than previously, where it was mainly the intended and unintended adverse effects on humans that have been studied. The fact that the CWAs are illicit weapons has hampered the development of standardized environmental results from the broader environmental toxicological community. ...
MODUM project continued the work on monitoring of the chemical weapons (CW) dumped in the Baltic Sea started in previous projects. As a new aspect, on board analysis methods – headspace gas chromatography-mass spectrometry (GC–MS) and capillary electrophoresis (CE) – were developed and tested in laboratory conditions and during cruises. The GC–MS method could be successfully applied on board to verify that collected sediment samples contained degradation products for sulfur mustard, one of the major chemical warfare agents dumped in Baltic Sea. This method could in future project be used during cruises to redirect sample collection in order to make most of the available ship time. Other part of the analysis task during MODUM project was the work done at the reach back laboratories. These analyses were done to both verify the results obtain on board and to fully identify the chemicals related to the sea-dumped CW agents. Reach back analysis of CW-related chemicals were done on sediment samples collected around a wreck in Bornholm Deep (same samples as analyzed on board) and on monitoring samples collected in Bornholm, Gotland and Gdańsk Deeps. The samples from Bornholm and Gotland Deeps are in line with previous findings. Samples from Gdańsk Deep are in line with previous findings that this area has been used as a dump site. Additionally, α-chloroacetophenone (CN) was found in the area for the first time. In addition to the analysis of CW-related chemicals, a new method was developed for measurement for arsenic concentrations in sediment samples. A method was also developed for arsenic speciation, which could help in estimation of the source of arsenic in the sediments.
... The environmental toxicity of Chemical warfare agents (CWAs) has in recent years received more attention (Della Torre et al. 2013;Sanderson et al. 2014) than previously, where it was mainly the intended and unintended adverse effects on humans that have been studied. The fact that the CWAs are illicit weapons has hampered the development of standardized environmental results from the broader environmental toxicological community. ...
This chapter summarizes the methods used within the MODUM project for monitoring chemical munition dumpsites. It includes general introduction to monitoring process, listing the requirements that are a basis for the establishment of full scale monitoring programme. It describes survey procedures, for locating dumped munitions, Sampling and analytical procedures for the detection of Chemical Warfare Agents, as well as the usage of fish as bioindicators are described. Modelling of pollutants originating from dumped munitions is presented and discussed. Only methods, which were proven to be most effective within the MODUM projects were selected, also data interpretation methods providing coherent information regarding the environmental risk are explained in details.
... The environmental toxicity of Chemical warfare agents (CWAs) has in recent years received more attention (Della Torre et al. 2013;Sanderson et al. 2014) than previously, where it was mainly the intended and unintended adverse effects on humans that have been studied. The fact that the CWAs are illicit weapons has hampered the development of standardized environmental results from the broader environmental toxicological community. ...
The environmental characteristics of the deep basins in the Baltic Sea and their impact on the occurrence of selected biota – benthos and fish communities – are described in chemical munitions dumping site areas. Results of the NATO-funded SfP project MODUM “Towards the Monitoring of Dumped Munitions Threat” (2013–2016) and other related previous activities regarding the impact of chemical warfare agents (CWA) on biodiversity and status of benthic fauna and regarding the health status of Baltic cod (Gadus morhua) are presented and discussed in the light of requirements for monitoring ecological risks associated with dumped CWA.
... The environmental toxicity of Chemical warfare agents (CWAs) has in recent years received more attention (Della Torre et al. 2013;Sanderson et al. 2014) than previously, where it was mainly the intended and unintended adverse effects on humans that have been studied. The fact that the CWAs are illicit weapons has hampered the development of standardized environmental results from the broader environmental toxicological community. ...
In this chapter, the data generated within MODUM and other related projects, i.e. CHEMSEA, MERCW and NordStream, contributing to the knowledge and data on occurrence, toxicity and effects of chemical warfare agents (CWAs) and their metabolites in the Baltic Sea, are aggregated. The data are evaluated and assessed in terms of risk quotients, and whether these point to risk or no-risk towards effects on lower tier organisms (e.g. algae and daphnia), and higher tier organisms (fish) from exposure to dumped CWAs and their metabolites in the study areas. To perform a semi-quantitative assessment of the environmental risk, a number of Lines of Evidences (LoEs) are set up, that take all aspects of the performed investigations into account. Each LoE is assigned +3, +2, +1, 0, −1, −2 or −3, indicating if the LoE is found to be for (+), against (−) or neutral (0) to an increased environmental risk of dumped CWAs. The weight also reflects the predictive power, or significance, of the individual LoEs. From nine LoEs a resulting summed score of +9 indicates that there is a weak to moderate potential for confirming the hypothesis. In order to qualify and increase the precision of the weights recommendations are given that can be addressed in future investigations regarding compounds, sites and species that could be in focus. Furthermore, recommendations for activities that will improve the exposure and toxicity data, that are inherent in the environmental risk assessment, are stated.
... The observed exposure concentrations presented in Table 2, combined with the field observations of e.g. Della Torre et al. (2010Torre et al. ( , 2013 and Baršienė et al. (2014Baršienė et al. ( , 2016 suggest that risk appraisal could still be improved primarily by more accurate chronic toxicity testing at higher tiers of the compounds detected in the environment. ...
AbstractAs a result of the disarmament of Germany after the Second World War, 65,000 tons of chemical munitions were dumped in the Baltic Sea. Approximately 13,000 tons containing chemical warfare agents (CWAs) of which 11,000 tons were dumped in the Bornholm Basin east of Bornholm. This paper addresses the ecotoxicity of compounds actually present in the Bornholm dumpsite by obtaining novel acute ecotoxicity data. EC50 values were successfully obtained for 12 CWAs from acute tests using Allivibrio fischeri (Microtox?). The three most toxic compounds were α-chloroacetophenone, 2-chlorovinylarsonic acid and 1,2,5-trithiepane having EC50 values of 11.20, 31.20 and 1170 ?g L?1, respectively. A. fischeri demonstrated hormesis when exposed to triphenylarsine and triphenylarsine oxide at concentrations of 100 and 50 mg L?1, respectively. Four different mixtures were assessed including compounds which were dissolvable; a mixture of sulphur mustard degradation products, a mixture of the three most toxic s
... Chemicals originating from warfare materials can eventually leak into the sea and spread from the sites of disposal over more distant areas. Leakage of toxic compounds from the corroded munitions has been recently suggested in dumpsites of the Baltic (Missiaen et al., 2010;Baršienė et al., 2014) and Adriatic Sea (Amato et al., 2006;Della Torre et al., 2013), and there are predictions that corrosion will lead to maximal leakage periods in the middle of the 21st century (Roose et al., 2011). Furthermore, the increasing demand for marine activities such as offshore wind farms and pipelines as well as changes in fishing practices raise new issues since these activities could also alter undisturbed munitions. ...
Anthropogenic contaminants reach the marine environment mostly directly from land-based sources, but there are cases in which they are emitted or re-mobilized in the marine environment itself. This paper reviews the literature, with a predominant focus on the European environment, to compile a list of contaminants potentially released into the sea from sea-based sources and provide an overview of their consideration under existing EU regulatory frameworks. The resulting list contains 276 substances and for some of them (22 antifouling biocides, 32 aquaculture medicinal products and 34 warfare agents) concentrations and toxicity data are additionally provided. The EU Marine Strategy Framework Directive Descriptor 8, together with the Water Framework Directive and the Regional Sea Conventions, provides the provisions against pollution of marine waters by chemical substances. This literature review should inform about the current state of knowledge regarding marine contaminant sources and provide support for setting-up of monitoring approaches, including hotspots screening.
... However, it should be stressed that only few publications, disclosing biological or ecological significance of chemical warfare agents exist (Sanderson et al., 2008Sanderson et al., , 2010 Della Torre et al., 2010 Baršienė et al., 2014). Attributed to the leakage of yperite from rusted bomb shells dumped in the southern Adriatic Sea, 3–4-times higher levels of As and Hg were recorded in fillets of two benthic fish species and genotoxicity effects of CWA were described (Della Torre et al., 2010 Torre et al., , 2013). The most significantly increased total geno-cytotoxicity levels were described in three fish species collected in 2009–2011 near a known chemical munition dumpsite in the Bornholm Basin. ...
... Gills are potentially in permanent contact with waterborne contaminants, which confers particular interest to this organ for genotoxicity assessment. For instance, Della Torre et al. (72) found elevated DNA strand breakage in the gills (but not liver, kidney, muscle or intestine) of conger eel collected from a chemical weapon dumping site at sea presumably contaminated by yperite (unquantified), which was interestingly, linked to increased histological lesions in the skin. In a more unusual approach, cryopreserved fish sperm has also been selected as target for the comet assay, albeit with moderate results (52,53). ...
Determining the genotoxic effects of pollutants has long been a priority in Environmental Risk
Assessment (ERA) for coastal ecosystems, especially of complex areas such as estuaries and
other confined waterbodies. The acknowledged link between DNA damage, mutagenicity and
carcinogenicity to the exposure to certain toxicants has been responsible to the growing interest in
determining the genotoxic effects of xenobiotics to wildlife as a measure of environmental risk. The
comet assay, although widely employed in in vivo and in vitro toxicology, still holds many constraints
in ERA, in large part owing to difficulties in obtaining conclusive cause–effect relationships from
complex environments. Nevertheless, these challenges do not hinder the attempts to apply the
alkaline comet assay on sentinel organisms, wild or subjected to bioassays in or ex situ (from fish
to molluscs) as well to standardise protocols and establish general guidelines to the interpretation
of findings. Fish have been regarded as an appealing subject due to the ease of performing the
comet assay in whole blood. However, the application of the comet assay is becoming increasingly
common in invertebrates (e.g. in molluscan haemocytes and solid tissues such as gills). Virtually all
sorts of results have been obtained from the application of the comet assay in ERA (null, positive
and inconclusive). However, it has become clear that interpreting DNA damage data from wild
organisms is particularly challenging due to their ability to adapt to continuous environmental
stressors, including toxicants. Also, the comet assay in non-model organisms for the purpose of
ERA implies different constraints, assumptions and interpretation of findings, compared with the
in vitro procedures from which most guidelines have been derived. This paper critically reviews
the application of the comet assay in ERA, focusing on target organisms and tissues; protocol
developments, case studies plus data handling and interpretation.
... Amato et al. [26] suggested additional fish biomarkers -but noted that causation needs to be established between the biomarker responses and CWA exposures. The work by Amato et al. [26] was expanded by Della Torre et al. [27], they also found indications of EROD activity impacts -but also that further causal elucidation was needed to definitively establish if the observations was a direct indicator of CWA exposure (in this case yperite). ...
... 33 While gills tissue appeared far more sensitive than liver, kidney, muscle and intestine of conger eel (Conger conger) sampled from a chemical weapons dumping area. 34 Previous mammalian studies have demonstrated that certain tissue types may have higher background levels of DNA damage due to presence of alkalisensitive sites in cells with highly condensed chromatin. 35 Similar studies comparing basal levels of DNA migration in mussel gill cells, haemocytes and fish erythrocytes using both mildly alkaline (pH 12.1) and alkaline versions (pH413) of Comet assay have supported this assumption. ...
Comet assay is a quick and versatile technique for assessing DNA damage in individual cells. It allows the detection of DNA single- and double-strand breaks, as well as the presence of alkali-labile sites and cross-links. Here we describe the protocols for the single-cell gel electrophoresis (Comet) assay in its alkaline (pH > 13), mild alkaline (pH = 12.1), and neutral (pH = 8) versions, when applied in marine animals.
The marine and coastal environment is a highly productive zone made up of several subsystems, including seagrass beds and coral reefs. It is a diverse habitat with a vast variety of living organisms that includes simple primitive to highly developed organisms. The famous marine ecosystems in which humans have invaded include Atlantic, Pacific, Indian, Arctic, and Antarctic Ocean basins. Since the origin of life on Earth, the seas have been responsible for preserving a healthy balance in the ecosystem of the entire planet. The waste products that are produced as a result of human activities eventually end the ocean ecosystem continuously. There are various chemical pollutants frequently drained in to the coastal and marine habitats. The ocean occupied a total area of about 360 million square kilometres, which is accounting for 71% of the Earth’s surface area. The most significant means of commercial transportation for import and export is sea route. The ocean along with the ocean ecosystem is a very significant part of our everyday lives. It can be said that the ocean has become the most important part of people’s life around the world. Hence, the state of the marine environment at the moment is not encouraging, and marine pollution has emerged as a major issue in modern civilisation. Moreover, the chemical and oil leakage incidents also affect the entire ecosystem. Thus, it is today’s urgent need to recover the maritime environment.
Marine environments are globally impacted by vast quantities of munition disposed following both World Wars. Dumped munitions contain conventional explosives, chemicals warfare agents as well as a variety of metals. Field monitoring studies around marine dumpsites report the presence of munition constituents in water and sediment samples. The growing interest and developments in the ocean as a new economic frontier underline the need to remediate existing dumpsites. Here, we provide a comprehensive assessment of the magnitude and potential risks associated with marine munition dumpsites. An overview of the global distribution of dumpsites identifying the most impacted areas is provided, followed by the currently available data on the detection of munition constituents in environmental samples and evidence of their toxic potential to human and environmental health. Finally, existing data gaps are identified and future research needs promoting better understanding of the impact of the dumped material on the marine environment suggested.
Recent studies have found primary degradation products of phenylarsenic chemical warfare agents (CWAs) accumulating in fish tissues, while the potential effects of these dumped phenylarsenic CWAs, such as Clark I and II, in the Baltic Sea biota are poorly understood. In this study, the metabolism and cytotoxicity of diphenylarsinic acid (DPA), a primary degradation product of phenylarsenic CWA, was studied by incubating rainbow trout cell line RTL-W1 cells in media with 100 mg/L DPA. Previously undescribed metabolites were identified by ultra-high performance liquid chromatography–high resolution mass spectrometry (UPHLC-HRMS). Moreover, the cytotoxicity of diphenylarsine glutathione conjugate (DPA-SG), the major metabolite of DPA, was studied. Cytotoxicity of the compounds was evaluated using the Neutral Red retention test (NRR), showing an IC50 value of 278 mg/L for DPA and 1.30 mg/L for DPA-SG, indicating that the glutathione (GSH) conjugate of DPA is more than two orders of magnitude toxic than DPA itself, suggesting that toxic properties of DPA are increased after conjugation with intracellular GSH leading enhanced toxicity after uptake. Results gained in this study give more detailed information for elucidating biological effects of dumped chemical munitions in marine environment. Moreover, the results help in assessing the environmental and health risks posed by marine munition continued presence and deterioration in the sea bottom.
This study represents the first attempt to assess genotoxicity and cytotoxicity effects in herring (Clupea harengus membras), flounder (Platichthys flesus), and cod (Gadus morhua callarias) caught at 47 study stations, located close to chemical munition dumpsites in the Gotland Basin, the Baltic Sea. Herring sampled from stations located in the center of chemical munition dumpsites exhibited the highest levels of micronuclei (MN) and total genotoxicity (ΣGentox), which is defined as the sum of frequencies of such nuclear abnormalities as micronuclei, nuclear buds, nuclear buds on the filament, and bi-nucleated erythrocytes with nucleoplasmic bridges. Exceptionally high and high ΣGentox risks were determined for flounder (89.47%), herring (79.31%), and cod (50%) caught at the stations located close to the chemical munition dumpsites.
The sinking of the cruise ship "Costa Concordia", which took place on January 13, 2012 off the Giglio island (Tuscany), also led to a remarkable environmental emergency with a worldwide echo. Set sail from the Civitavecchia harbour towards Savona, the large ship, following a incautious manoeuvre to approach the coast of Giglio Island, at 21:45 hit at high speed a submerged rocks called " Le Scole ”. This volume outlines, in particular, the activities that the researchers, technologists and technicians of ISPRA/SNPA supported in the management of the "Concordia" emergency and highlights the environmental controls carried out as well as the advices and indications provided by them to the institutions responsible for coordinating the interventions, to reduce, as far as possible, the environmental consequences of the shipwrecking.
Recently, sea-dumped chemical weapons (CWs) containing toxic chemical warfare agents (CWAs) have raised international attention. It is well known that CWAs are leaking from corroded munitions causing a risk to the surrounding marine environment, while the impact on marine biota is still unknown. In this study, cod (Gadus morhua) was used as a model species to study the possible bioaccumulation of phenylarsenic CWAs and their negative effects at multiple levels of biological organization on fish living in the vicinity of a major CWs dumpsite in the Bornholm Basin in the Baltic Sea. In total, 14% of the cod muscle samples collected close to the main dumpsite contained trace levels of phenylarsenic CWAs. However, most of the biomarkers measured did not show clear differences between this area compared with a lesser contaminated reference area. On the other hand, significant changes in some biomarkers were observed in individuals containing trace levels of CWA-related chemicals. The results gained in this study have significant importance for environmental risk assessment and for evaluating the risk of CWA contamination for human seafood consumers.
The sea bottom of the Skagerrak Strait (North Sea) contains munitions loaded with chemical warfare agents (CWA), mostly stored in shipwrecks scuttled intentionally after the end of the World War II. The munition shells inside the wrecks are in different states of deterioration and corrosion and their environmental risk potential is unknown. The Atlantic hagfish (Myxine glutinosa), a sediment-dwelling chordate, was used as a model organism to study the potential impact of dumped CWA on the local ecosystem by using biochemical biomarkers. The hagfish were collected in 2017 and 2018 at three sampling sites: in the immediate vicinity of a wreck with CWA in the Skagerrak, a few kilometres from the wreck, and a reference site 21 km from the wreck, considered to be free of CWA. Significant differences were observed between the wreck site and the reference sites in the activities of glutathione reductase, superoxide dismutase and glutathione S-transferase, while the activity levels of catalase and acetylcholinesterase were identical at all sites. The recorded differences demonstrated negative biological effects in the hagfish sampled close to the dumped chemical munitions. Due to the limited knowledge of hagfish biology and of the extent of CWA contamination in Skagerrak, the results presented here warrant more research to further elucidate the potential environmental risks of the scuttled wrecks. The usefulness of the species as a bioindicator organism is further discussed.
Within the framework of the international project DAIMON (Decision Aid for Marine Munitions), the impact of dumped chemical munitions on fish health was investigated. The Skagerrak Straight (North Sea, at 600 m depth) contains munitions with chemical warfare agents (CWA), scuttled after the end of World War II. Studies of liver histopathology in Atlantic hagfish (Myxine glutinosa) were carried out at three sampling sites: at a wreck with CWA in the Skagerrak (n = 82), a Skagerrak reference site considered to be free of CWA (n = 14) and at a reference site in the northern North Sea outside the Skagerrak (n = 17). Liver lesions were diagnosed and categorized according to standardized ICES and BEQUALM protocols and OSPAR guidelines.
Non-specific liver lesions were found in 87.6% of 113 hagfish examined. The prevalence of pre-neoplastic lesions was 7.1% and of neoplastic lesions 6.2%. There was no statistically significant difference in prevalence between hagfish samples from the wreck site and from the reference site near the wrecks. However, at the reference site in the northern North Sea, the prevalence of non-specific lesions was low and neither pre-neoplastic nor neoplastic lesions were observed.
Eight nuclear abnormalities of genotoxicity and cytotoxicity were studied in peripheral blood erythrocytes of herring (Clupea harengus membras), flounder (Platichthys flesus), and Atlantic cod (Gadus morhua) sampled (2010–2017) from the Polish and the Lithuanian Exclusive Economic Zones (EEZ) in the Baltic Sea. At all study stations, total genotoxicity (∑Gentox) was found to be higher than total cytotoxicity (∑Cytox). A significant time-related decrease in genotoxicity was detected in the Lithuanian EEZ (2015–2017), while in the Polish EEZ (2014–2016), the opposite tendency was revealed. The highest ∑Gentox and ∑Cytox values recorded in fish sampled at the study stations located relatively close to each other clearly indicate an increased environmental genotoxicity and cytotoxicity pressure for fish in these areas. Exceptionally high and high-level genotoxicity risks to herring followed by those to flounder and cod were determined at a higher percentage of the stations studied.
Baltic blue mussels (Mytilus trossulus) were implemented to assess potential toxicity, health impairments and bioaccumulation of dumped chemical warfare agents on marine benthic organisms. Mussels were collected from a pristine cultivation side and exposed under laboratory conditions to different mixtures of chemical warfare agents (CWAs) related phenyl arsenic compounds, Clark I and Adamsite as well as chloroacetophenone. Using a multi-biomarker approach, mussels were assessed thereafter for effects at different organisational levels ranging from geno-to cytotoxic effects, differences in enzyme kinetics and immunological responses. In an integrated approach, chemical analysis of water and tissue of the test organisms was performed in parallel. The results show clearly that exposed mussels bioaccumulate the oxidized forms of chemical warfare agents Clark I, Adamsite (DAox and DMox) and, to a certain extent, also chloroacetophenone into their tissues. Adverse effects in the test organisms at subcellular and functional level, including cytotoxic, immunotoxic and oxidative stress effects were visible. These acute effects occurred even at the lowest test concentration.
This chapter reviews the environmental toxicity of CWAs and their metabolites as well as mixtures of CWAs. We used Microtox™ to generate EC50 value for 11 compounds. We observed hormetic effects for two compounds namely Triphenylarsine and Triphenylarsine oxide. None of the mixtures tested show sign of synergism. Two compounds can be characterized as very toxic as both α-chloroacetophenone (EC50 = 11.20 μg L⁻¹) and 2-chlorovinylarsinic acid (EC50 = 31.20 μg L⁻¹) demonstrated EC50 values below 1000 μg L⁻¹. Several compounds can be characterized as toxic as 1,2,5-trithiepane (EC50 = 1170 μg L⁻¹), 1,4,5-oxadithiepane (EC50 = 1700 μg L⁻¹), phenarsazinic acid (EC50 = 5330 μg L⁻¹) and 1,4-dithiane (EC50 = 9970 μg L⁻¹) as these compounds demonstrated EC50 values between 1000 μg L⁻¹ and 10,000 μg L⁻¹. An D. magna acute LC50 for, the compound most frequently detected compound (DPA [ox]), was determined to be 100,000 μg L⁻¹. A chronic D. magna LC5019days of 640 μg L⁻¹ was derived for the compound. A 14-day locomotor behaviour test on adult male Zebrafish (Danio rerio) revealed altered behaviour when exposed to concentrations of 1,4,5-oxadithiepane down to 40.3 ± 2.9 μg L⁻¹. A NOECweight and NOECmortality greater than 1533 μg L⁻¹ was determined for 1,4,5-oxadithiepane.
Concerns about the adverse effects of chemicals present in the environment have created a need for better systems to assess their potential consequences on human health. One potential solution is the versatile and state-of-the-art Comet assay. Simple, sensitive, rapid and visual, this modern toxicological method allows quantitative and qualitative assessment of DNA damage in single cells. This assay is used in diverse fields ranging from clinical applications, human monitoring and environmental toxicology through to genetic toxicity testing.
This updated and revised edition of The Comet Assay in Toxicology provides the latest information on this important tool. It addresses, in-depth, the different protocols, statistical analyses and applications used worldwide. It also includes the guidelines recommended by the Working Group on Comet Assay. The book begins with a review of the genesis of the assay for those new to the technique and goes on to explain procedures followed to assess different types of DNA damage, various applications of the assay, and guidelines for the conduct of the assay in in vitro and in vivo systems. New chapters written for this edition will provide information on the most contemporary approaches and applications, including in silico approaches, on meta-analysis of data and on the application of the Comet Assay in nanotoxicology.
This book will serve as both a reference and a guide to students as well as investigators in the biomedical, biochemical and pharmaceutical sciences fields.
Sulfur mustard (SM) and similar bifunctional agents have been used as chemical weapons for almost 100 years. Victims of high-dose exposure, both combatants and civilians, may die within hours or weeks, but low-dose exposure causes both acute injury to the eyes, skin, respiratory tract and other parts of the body, and chronic sequelae in these organs are often debilitating and have a serious impact on quality of life. Ever since they were first used in warfare in 1917, SM and other mustard agents have been the subjects of intensive research, and their chemistry, pharmacokinetics and mechanisms of toxic action are now fairly well understood. In the present article we review this knowledge and relate the molecular-biological basis of SM toxicity, as far as it has been elucidated, to the pathological effects on exposure victims.
The utility of splenic macrophage aggregates (MAs) as an indicator of fish exposure to degraded environments was evaluated in several species of estuarine fishes as part of the Environmental Protection Agency's Environmental Monitoring and Assessment Program–Estuaries (EMAP-E). Using image analysis, we measured the number and mean size of MAs per square millimeter on tissue sections of spleen from 983 fishes representing seven species from 266 stations scattered across coastal estuaries of the Gulf of Mexico. At 16 stations, at least one fish exhibited a high density of MAs (>40 MAs/mm). Densities of MAs that exceeded 40/mm correlated with exposure to either hypoxic conditions or sediment contamination. Fisher's exact test showed that the observed frequencies of joint occurrence between high numbers of MAs and both high sediment contaminants and low dissolved oxygen were significantly greater than the expected background frequencies. For all 16 sites where MAs were greater than 40/mm, sediments displayed at least one contaminant at a concentration in the highest 5% of those observed for all Gulf of Mexico stations. Additionally, comparison of subjective visual analyses with the image analysis measurements showed a strong correlation, indicating that similar analyses can be performed without computer image analysis. This study demonstrates that splenic MAs are effective biotic indicators for discriminating between fish exposed to degraded and nondegraded environments.
In order to gain preliminary knowledge about the threat to marine ecosystems due to leakage of chemical warfare agents (CWAs)
and other pollutants from rusting bombshells on the seabed, a case study was conducted in a dumping area in the southern Adriatic
Sea (depth 200–300m). Following electroacoustic and magnetometric surveys of the CWA dumping area, an integrated ecotoxicological
approach was used. This approach was based on analysis of CWA residues and their metabolites, including arsenic, in sediment
and organisms, as well as multimarker methodology including the Health Assessment Index, histological lesion analysis and
enzyme assays. Two sentinel species were selected, the blackbelly rosefish [Helicolenus dactylopterus (Delaroche, 1809)] and the European conger (Conger conger L., 1758). Sediment analysis revealed the presence of CWA degradation products, including 1-4-thioxane and 1-4-dithiane.
Tissues of fish from the CWA dumping site showed higher levels of arsenic than those from the reference site. Neither CWAs
nor their metabolites were detected in fish tissues. Arsenic levels recorded in blackbelly rosefish were well above those
reported for other fish species from the southern Adriatic, much higher than the FDA limit for food (2.6mgkg−1) and close to the LD50 calculated for mammals (20mgkg−1 body weight). The presence of pollutants in the CWA dumping site was also confirmed by pathological lesions in both species
and EROD activity, two to three times higher than in fish from the reference site (16.45±8.08 and 8.05±5.87pmolmin−1mg protein−1 in blackbelly rosefish and 269±24.92 and 78.71pmolmin−1mgprotein−1 in European conger, respectively). Cholinesterase activity seemed unaffected in muscle of both species, whereas in brain
they were one-third of those recorded in fish from the reference site (14.22±10.05 and 72.87nmolmin−1mgprotein−1, respectively). This suggests that acetylcholinesterase is sensitive to CWAs. In conclusion, the agreement of all the chemical
and biological parameters investigated suggests that the integrated ecotoxicological approach used is appropriate to reveal
the presence and biological effects of CWAs in the marine ecosystem.
Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 μM) at 4 °C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
In this review, a wide array of bioaccumulation markers and biomarkers, used to demonstrate exposure to and effects of environmental contaminants, has been discussed in relation to their feasibility in environmental risk assessment (ERA). Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as bioconcentrators to identify certain substances with low water levels and to assess exposure of aquatic organisms. Since it is virtually impossible to predict the fate of xenobiotic substances with simple partitioning models, the complexity of bioaccumulation should be considered, including toxicokinetics, metabolism, biota-sediment accumulation factors (BSAFs), organ-specific bioaccumulation and bound residues. Since it remains hard to accurately predict bioaccumulation in fish, even with highly sophisticated models, analyses of tissue levels are required. The most promising fish bioaccumulation markers are body burdens of persistent organic pollutants, like PCBs and DDTs. Since PCDD and PCDF levels in fish tissues are very low as compared with the sediment levels, their value as bioaccumulation markers remains questionable. Easily biodegradable compounds, such as PAHs and chlorinated phenols, do not tend to accumulate in fish tissues in quantities that reflect the exposure. Semipermeable membrane devices (SPMDs) have been successfully used to mimic bioaccumulation of hydrophobic organic substances in aquatic organisms. In order to assess exposure to or effects of environmental pollutants on aquatic ecosystems, the following suite of fish biomarkers may be examined: biotransformation enzymes (phase I and II), oxidative stress parameters, biotransformation products, stress proteins, metallothioneins (MTs), MXR proteins, hematological parameters, immunological parameters, reproductive and endocrine parameters, genotoxic parameters, neuromuscular parameters, physiological, histological and morphological parameters. All fish biomarkers are evaluated for their potential use in ERA programs, based upon six criteria that have been proposed in the present paper. This evaluation demonstrates that phase I enzymes (e.g. hepatic EROD and CYP1A), biotransformation products (e.g. biliary PAH metabolites), reproductive parameters (e.g. plasma VTG) and genotoxic parameters (e.g. hepatic DNA adducts) are currently the most valuable fish biomarkers for ERA. The use of biomonitoring methods in the control strategies for chemical pollution has several advantages over chemical monitoring. Many of the biological measurements form the only way of integrating effects on a large number of individual and interactive processes in aquatic organisms. Moreover, biological and biochemical effects may link the bioavailability of the compounds of interest with their concentration at target organs and intrinsic toxicity. The limitations of biomonitoring, such as confounding factors that are not related to pollution, should be carefully considered when interpreting biomarker data. Based upon this overview there is little doubt that measurements of bioaccumulation and biomarker responses in fish from contaminated sites offer great promises for providing information that can contribute to environmental monitoring programs designed for various aspects of ERA.
Understanding the ecotoxicological effects of arsenic in the environment is paramount to mitigating its deleterious effects on ecological and human health, particularly on the immune response. Toxicological and long-term health effects of arsenic exposure have been well studied. Its specific effects on immune function, however, are less well understood. Eukaryotic immune function often includes both general (innate) as well as specific (adaptive) responses to pathogens. Innate immunity is thought to be the primary defense during early embryonic development, subsequently potentiating adaptive immunity in jawed vertebrates, whereas all other eukaryotes must rely solely on the innate immune response throughout their life cycle. Here, we review the known ecotoxicological effects of arsenic on general health, including immune function, and propose the adoption of zebrafish as a vertebrate model for studying such effects on innate immunity.
Sulfur mustard (2,2-dichlorodiethyl sulfide, SM) is one of the vesicant classes of chemical warfare agents that causes blistering in the skin and mucous membranes, where it can have lingering long-term effects for up to ten years (1). SM was employed extensively by the Iraqi army against not only Iranian soldiers but also civilians between 1983 and 1988, resulting in over 100,000 chemical casualties. Approximately 45,000 victims are still suffering from long-term effects of exposure (2,3). More than 90% of the patients exposed to SM exhibit various cutaneous lesions in the affected area. The human skin can absorb approximately 20% of the SM through exposure. Up to 70% of the chemical is concentrated in the epidermis and the remainder in the basement membrane and in the dermis (4).Sulfur mustard exists in different physical states. The liquid form of SM evaporates slowly in cold weather and can penetrate through the clothing, thereby increasing exposure. However, the gas form readily diffuses in the air and it can be inhaled, leading to systemic absorption. In addition, warm temperatures are ideal conditions that liquid SM present in the clothing of the exposed individual could be converted to gas form. SM-induced clinical cutaneous symptoms include itching and burning. Other clinical findings include erythema or painless sunburn, bulla, hypo- and hyper pigmentation in both exposed and unexposed areas (5,6) The mechanism and biochemical cascade of SM-induced cutaneous manifestations are not completely understood but several published pathways support many of the know facts. Our current understanding fails to explain the time interval between the acute chemical exposure and the late-onset and delayed tissue damage (7,8). The aim of this article is to review the acute and long-term cutaneous findings resulting from SM exposure. Also, cellular and molecular mechanism involved in SM-induced skin pathology have been discussed.
Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.
Sulfur mustard (HD) is an alkylating and cytotoxic chemical warfare agent, which inflicts severe skin toxicity and an inflammatory response. Effective medical countermeasures against HD-caused skin toxicity are lacking due to limited knowledge of related mechanisms, which is mainly attributed to the requirement of more applicable and efficient animal skin toxicity models. Using a less toxic analog of HD, chloroethyl ethyl sulfide (CEES), we identified quantifiable inflammatory biomarkers of CEES-induced skin injury in dose- (0.05-2 mg) and time- (3-168 h) response experiments, and developed a CEES-induced skin toxicity SKH-1 hairless mouse model. Topical CEES treatment at high doses caused a significant dose-dependent increase in skin bi-fold thickness indicating edema. Histopathological evaluation of CEES-treated skin sections revealed increases in epidermal and dermal thickness, number of pyknotic basal keratinocytes, dermal capillaries, neutrophils, macrophages, mast cells, and desquamation of epidermis. CEES-induced dose-dependent increases in epidermal cell apoptosis and basal cell proliferation were demonstrated by the terminal deoxynucleotidyl transferase (tdt)-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen stainings, respectively. Following an increase in the mast cells, myeloperoxidase activity in the inflamed skin peaked at 24 h after CEES exposure coinciding with neutrophil infiltration. F4/80 staining of skin integuments revealed an increase in the number of macrophages after 24 h of CEES exposure. In conclusion, these results establish CEES-induced quantifiable inflammatory biomarkers in a more applicable and efficient SKH-1 hairless mouse model, which could be valuable for agent efficacy studies to develop potential prophylactic and therapeutic interventions for HD-induced skin toxicity.
Ethoxyresorufin (7-ethoxyphenoxazone) has been synthesized to serve as a model substrate for the simple, direct, fluorimetric assay of the hepatic microsomal O-dealkylation reaction. Liver microsomes from rat and hamster O-deethylated ethoxyresorufin to resorufin (7-hydroxyphenoxazone) in an NADPH and oxygen dependent reaction involving the flavoprotein, NADPH cytochrome c reductase, and cytochrome P-450. The reaction was performed in a fluorimeter cuvette and monitored directly by recording the increase in fluorescence associated with the formation of resorufin. The reaction exhibited a relatively low apparent K(m) which was different in the control and induced rat (150-259 nM) and hamster (40-163 nM). Phenobarbital, but not 3-methylcholanthrene, pretreatment altered the apparent K(m) for ethoxyresorufin deethylation observed with rat liver microsomes, whereas both inducing agents increased the apparent K(m) for the reaction with hamster liver microsomes. 3 Methylcholanthrene pretreatment of the animals increased the reaction apparent V(max) 70-fold in the rat and 8-fold in the hamster, whereas phenobarbital pretreatment did not stimulate the reaction in either species.
1 Sulphur mustard reacts directly with benzenethiols and cysteine esters in aqueous medium.
2 Benzenethiols diffuse into lung slices in short term culture.
3 Treatment of lung slices in short term culture with benzenethiols does not protect cellular glutathione from conjugation with sulphur mustard.
4 Following uptake of cysteine ester into lung slices cysteine is elevated but this does not protect cellular glutathione from sulphur mustard.
We include in this review an assessment of the formation, environmental fate, and mammalian and ecotoxicity of CW agent degradation products relevant to environmental and occupational health. These parent CW agents include several vesicants: sulfur mustards [undistilled sulfur mustard (H), sulfur mustard (HD), and an HD/agent T mixture (HT)]; nitrogen mustards [ethylbis(2-chloroethyl)amine (HN1), methylbis(2-chloroethyl)amine (HN2), tris(2-chloroethyl)amine (HN3)], and Lewisite; four nerve agents (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX), tabun (GA), sarin (GB), and soman (GD)); and the blood agent cyanogen chloride. The degradation processes considered here include hydrolysis, microbial degradation, oxidation, and photolysis. We also briefly address decontamination but not combustion processes. Because CW agents are generally not considered very persistent, certain degradation products of significant persistence, even those that are not particularly toxic, may indicate previous CW agent presence or that degradation has occurred. Of those products for which there are data on both environmental fate and toxicity, only a few are both environmentally persistent and highly toxic. Major degradation products estimated to be of significant persistence (weeks to years) include thiodiglycol for HD; Lewisite oxide for Lewisite; and ethyl methyl phosphonic acid, methyl phosphonic acid, and possibly S-(2-diisopropylaminoethyl) methylphosphonothioic acid (EA 2192) for VX. Methyl phosphonic acid is also the ultimate hydrolysis product of both GB and GD. The GB product, isopropyl methylphosphonic acid, and a closely related contaminant of GB, diisopropyl methylphosphonate, are also persistent. Of all of these compounds, only Lewisite oxide and EA 2192 possess high mammalian toxicity. Unlike other CW agents, sulfur mustard agents (e.g., HD) are somewhat persistent; therefore, sites or conditions involving potential HD contamination should include an evaluation of both the agent and thiodiglycol.
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The fluorescent compound 4-methylumbelliferone (4MU) can be used to detect uridine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a mean V(max) of 19.9 nmol/min/mg of protein and a mean K(m) of 652.5 microM on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V(max) was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P <.0001) higher than for the extraction method. The mean K(m), 175. 4 +/- 24.2 microM (CV = 14.5%), was significantly lower (P <.0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% for V(max) and 8 and 35% for K(m), respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2. 6 nmol/min/mg of protein, respectively, at 200 microM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.
In the Baltic Sea has appeared a great amount of fishes on which skin observed many anatomicopathological changes of different kind. It caused a realization of the following investiga tions: parasitological, virological, histological, microbiological and chemical on the presence of havy metal salts, pesticiede and arsenie as a remains of yperite disintegration. The investigations have proved that the direct agents causing diseases in marine environment pollution and the presence of a great amount of bacteria of the genus Areomonas, Pseudomonas, Vibrio, Proteus.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The health assessment index (HAI) is an extension and refinement of a previously published field necropsy system. The HAI is a quantitative index that allows statistical comparisons offish health among data sets. Index variables are assigned numerical values based on the degree of severity or damage incurred by an organ or tissue from environmental stressors. This approach has been used to evaluate the general health status offish populations in a wide range of reservoir types in the Tennessee River basin (North Carolina, Tennessee, Alabama, Kentucky), in Hartwell Reservoir (Georgia, South Carolina) that is contaminated by polychlorinated biphenyls, and in the Pigeon River (Tennessee, North Carolina) that receives effluents from a bleached kraft mill. The ability of the HAI to accurately characterize the health offish in these systems was evaluated by comparing this index to other types of fish health measures (contaminant, bioindicator, and reproductive analysis) made at the same time as the HAI. In all cases, the HAI demonstrated the same pattern offish health status between sites as did each of the other more sophisticated health assessment methods. The HAI has proven to be a simple and inexpensive means of rapidly assessing general fish health in field situations.
Skin ulcers on fish are one of the most well-recognized indicators of polluted or otherwise stressed aquatic environments. In recent years, skin ulcer epidemics have been either experimentally or epidemiologically linked to exposure to a number of xenobiotic chemicals as well as to biotoxins. Some of these agents, such as toxins produced by the dinoflagellate alga Pfiesteria, have led to serious concerns about the health of aquatic ecosystems, such as estuaries along the east coast of the United States. However, a number of other risk factors besides Pfiesteria have been shown to damage epithelium and may also play important roles in skin ulcer pathogenesis. In addition, increasing evidence indicates that not only may skin damage occur via direct contact with toxins, but it may also be induced indirectly from physiological changes that result from exposure not only to toxins but also to other environmental stressors, such as pH and temperature extremes. The multifactorial pathways that operate at both the ecological and the organismal levels as well as the nonspecific response of the skin to insults make it very challenging to link epidemic skin ulcers to any single cause in natural aquatic populations. Consequently, using pathology to unequivocally identify the specific cause of a lesion (eg, Pfiesteria exposure) is not a valid approach. Only with an increased understanding of the basic mechanisms leading to skin damage (including development of specific biomarkers for specific toxins), along with a better understanding of ecological processes operating in these environments, will we be able to discern the relative importance of various risk factors in skin ulcer development.
A study of teleost (bony fish) epidermis by light and electron microscopy has demonstrated that this epidermis consists predominantly of filament-containing cells. In comparing teleost epidermis with that of mammals, the absence of a cell sequence comparable to basal, spinous, granular, and horny cells is immediately apparent.In filament-containing cells most organelles cluster around the centrally placed nucleus and 70 Å diameter filaments course through the peripheral cytoplasm. Shorter and less dense filamentous wisps and ribosomes are seen between the filaments and central organelle complex. These filamentous wisps are thought to represent precursors of the more peripheral filaments. Filament-containing cells in the basal and mid-layers of the epidermis are structurally similar. Differentiation of these cells is apparent only in the surface layer where microvilli develop which are covered by a fuzz layer; a terminal web is formed, an overall increase in protoplasmic density occurs, and some organelles undergo structural changes.
In connection with installation of two natural gas pipelines through the Baltic Sea between Russia and Germany, there has been concern regarding potential re-suspension of historically dumped chemical warfare agents (CWA) in a nearby dump site and the potential environmental risks associated. 192 sediment and 11 porewater samples were analyzed for CWA residues, both parent and metabolites in 2008 and 2010 along the pipeline corridor next to the dump site. Macrozoobenthos and background variables were also collected and compared to the observed CWA levels and predicted potential risks. Detection frequencies and levels of intact CWA found were low, whereas CWA metabolites were more frequently found. Re-suspension of CWA residue-containing sediment from installation of the pipelines contributes marginally to the overall background CWA residue exposure and risk along the pipeline route. The multivariate weight-of-evidence analysis showed that physical and background parameters of the sediment were of higher importance for the biota than observed CWA levels.
Sulfur mustard (SM, bis(2-chloroethyl)sulfide) is a bifunctional alkylating agent that causes dermal inflammation, edema and blistering. To investigate the pathogenesis of SM-induced injury, we used a vapor cup model which provides an occlusive environment in which SM is in constant contact with the skin. The dorsal skin of SKH-1 hairless mice was exposed to saturated SM vapor or air control. Histopathological changes, inflammatory markers and DNA damage were analyzed 1-14 days later. After 1 day, SM caused epidermal thinning, stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Cleaved caspase-3 and phosphorylated histone 2A.X (phospho-H2A.X), markers of apoptosis and DNA damage, respectively, were increased whereas proliferating cell nuclear antigen (PCNA) was down-regulated after SM exposure. By 3 days, epithelial cell hypertrophy, edema, parakeratosis and loss of epidermal structures were noted. Enzymes generating pro-inflammatory mediators including myeloperoxidase and cyclooxygenase-2 were upregulated. After 7 days, keratin-10, a differentiation marker, was evident in the stratum corneum. This was associated with an underlying eschar, as neoepidermis began to migrate at the wound edges. Trichrome staining revealed increased collagen deposition in the dermis. PCNA expression in the epidermis was correlated with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, there was epidermal regeneration with extensive hyperplasia, and reduced expression of cleaved caspase-3, cyclooxygenase-2 and phospho-H2A.X. These findings are consistent with the pathophysiology of SM-induced skin injury in humans suggesting that the hairless mouse can be used to investigate the dermatoxicity of vesicants and the potential efficacy of countermeasures.
Sulfur mustard (SM), a vessicating agent, has been used in chemical warfare since 1918. The purpose of this study was to quantitate SM vapor deposition, tissue distribution, and excretion following intratracheal inhalation in rats and cutaneous exposure in guinea pigs. 14C-SM vapors for inhalation studies were generated by metering liquid 14C-SM into a heated J tube. Vapors were transported via carrier air supplemented with oxygen and isoflurane to an exposure plenum. Anesthetized rats with transorally placed tracheal catheters were connected to the plenum port via the catheter hub for exposure (approximately 250 mg 14C-SM vapor/m(3); 10 min). For dermal exposure, 3 Teflon cups (6.6 cm(2) exposure area per cup) were applied to the backs of each animal and vapors (525 mg 14C-SM/m(3); 12 min) were generated by applying 6 μl 14C-SM to filter paper within each cup. Animals were euthanized at selected times up to 7 d postexposure. SM equivalents deposited in rats and guinea pigs were 18.1 ± 3 μg and 29.8 ± 5.31 μg, respectively. Inhaled SM equivalents rapidly distributed throughout the body within 2 h postexposure, with the majority (>70%) of material at that time located in carcass and pelt. In guinea pigs, >90% of deposited SM equivalents remained in skin, with minor distribution to blood and kidneys. Urine was the primary route of excretion for both species. Results indicate inhaled SM is rapidly absorbed from the lung and distributed throughout the body while there is limited systemic distribution following cutaneous exposure.
The objective of these studies was to provide detailed analyses of the time course of sulfur mustard (SM) vapor-induced clinical, histological, and biochemical changes following cutaneous exposure in hairless guinea-pigs. Three 6 cm(2) sites on the backs of each guinea-pig were exposed to SM vapor (314 mg(3) ) for 6 minutes (low dose) or 12 minutes (high dose). Animals were killed at 6, 24, and 48 hours, or 2 weeks postexposure. Erythema, edema, histopathology, and analysis of matrix metalloproteinase (MMP)-2 and -9 content were evaluated. Erythema was observed by 6 hours, and edema by 24 hours postexposure. Vapor exposure caused epidermal necrosis with varying degrees of dermatitis, ulceration, hemorrhage, and separation of the dermis from the epidermis. Later changes included epidermal regeneration with hyperplasia and formation of granulation tissue in the dermis with loss of hair follicles and glandular structures. Relative amounts of pro and active MMP-2 and MMP-9 were significantly increased in the high-dose SM group at 2 weeks. Erythema, edema, and histologic changes are consistent with findings among human victims of SM attack. This model, with observations to 2 weeks, will be useful in assessing the efficacy of countermeasures against SM.
Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE₂ synthases, leukotriene (LT) A₄ hydrolase and LTC₄ synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.
Does the post-WWII burial at sea of chemical weapons still pose a human and environmental risk?
Aim of this study was to evaluate the responsiveness of red mullet (Mullus barbatus) liver detoxification enzymes to PAHs at transcriptional and post-transcriptional levels in the field. Fish were captured in the north-eastern Adriatic Sea, close to an oil refinery. Sixteen PAHs (EPA) were determined in sediments and fish fillets; transcription levels of cyp1a, cyp3a and abcc2 genes and EROD, BROD, B(a)PMO, BFCOD, GST and UDPGT enzymatic activities were measured. Levels of PAHs in sediments reflect the oil pollution gradient of the area, with weak correspondence in fish fillets. cyp1a gene transcription and EROD, B(a)PMO and BFCOD activities were significantly induced in the oil refinery site, and a slight up-regulation of cyp3a and abcc2 was also observed. GST and UDPGT remained unchanged. The present study provides the first data on detoxification responses at transcriptional levels in the liver of red mullet and confirms phase I enzymes as suitable biomarkers of exposure to PAHs in field studies.
Background/purpose: Skin exposure to sulfur mustard (HD) results in erythema, edema and severe injury, which take long time to heal and might impose a heavy burden on the health system. Despite many years of research, there is no treatment that prevents the development of the cytotoxic effects of HD causing acute and prolonged damage to the skin. Therefore, it is of great importance to develop treatments that will ameliorate the extent of injury and improve as well as shorten the healing process. The aim of the present study was to establish a small animal model for a long-term HD-induced skin injury using the hairless guinea-pig (HGP) and to further test the efficacy of anti-inflammatories in ameliorating the pathology.
The aim of the present study was to evaluate the environmental threat to benthic species from chemical weapons dumped in the southern Adriatic Sea. An ecotoxicological approach using chemical analysis and biological responses was applied, in two sentinel species: the Blackbelly rosefish Helicolenus dactylopterus and European conger Conger conger. Specimen were collected in a stretch of sea, where had been dumped war materials and from a reference site free of ordnance. Residues of yperite, Hg and As were measured in fish fillets. Skin, liver, kidney and spleen were examined for histopathological and macroscopical lesions. Liver detoxifying capacities (EROD and UDPGT) and genotoxicity (comet assay) were also investigated. As and Hg levels were three-four times higher than those from the reference site in both species (p<0.001). Both species captured in dumping site showed clear signs of chronic illness according to the health assessment index (HAI). Deep ulcers and nodules were observed on skin and external organs. Histological lesions such as periportal and bile duct fibrosis, pericholangitis, steatosis, granuloma and elevated splenic MMCs were detected in liver and spleen. Significantly higher EROD activities were also found in both species from dumping site (p<0.01). Comet assay revealed genotoxicty in gills of C. conger from dumping site, indicating uptake of chemical warfare agents through fish gills. European conger was found to be a more sensitive bioindicator of this type of contamination than the Blackbelly rosefish.
Sulfur mustard (SM) is a strong alkylating agent, which produces subepidermal blisters, erythema and inflammation after skin contact. Despite the well-described SM-induced gross and histopathological changes, the exact underlying molecular mechanisms of these events are still a matter of research. As part of an international effort to elucidate the components of cellular signal transduction pathways, a large body of data has been accumulated in the last decade of SM research, revealing deeper insight into SM-induced inflammation, DNA damage response, cell death signaling, and wound healing. SM potentially alkylates nearly every constituent of the cell, leading to impaired cellular functions. However, SM-induced DNA alkylation has been identified as a major trigger of apoptosis. This includes monofunctional SM-DNA adducts as well as DNA crosslinks. As a consequence, DNA replication is blocked, which leads to cell cycle arrest and DNA single and double strand breaks. The SM-induced DNA damage results in poly(ADP-ribose) polymerase (PARP) activation. High SM concentrations induce PARP overactivation, thus depleting cellular NAD(+) and ATP levels, which in consequence results in necrotic cell death. Mild PARP activation does not disturb cellular energy levels and allows apoptotic cell death or recovery to occur. SM-induced apoptosis has been linked both to the extrinsic (death receptor, Fas) and intrinsic (mitochondrial) pathway. Additionally, SM upregulates many inflammatory mediators including interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha) and others. Recently, several investigators linked NF-kappaB activation to this inflammatory response. This review briefly summarizes the skin toxicity of SM, its proposed toxicodynamic actions and strategies for the development of improved medical therapy.
The present study was aimed at elucidating the effect of chronic low-level arsenic exposure on the head kidney (HK) of Clarias batrachus and at determining the changes in head kidney macrophage (HKM) activity in response to arsenic exposure. Chronic exposure (30 days) to arsenic (As(2)O(3), 0.50 microM) led to significant increase in arsenic content in the HK accompanied by reduction in both HKM number and head kidney somatic index (HKSI). Arsenic induced HK hypertrophy, reduction in melano-macrophage population and increased hemosiderin accumulation. Transmission electron microscopy of 30 days exposed HKM revealed prominent endoplasmic reticulum, chromatin condensation and loss in structural integrity of nuclear membrane. Head kidney macrophages from exposed fish demonstrated significant levels of superoxide anions but on infection with Aeromonas hydrophila were unable to clear the intracellular bacteria and died. Exposure-challenge experiments with A. hydrophila revealed that chronic exposure to micromolar concentration of arsenic interfered with the phagocytic potential of HKM, helped in intracellular survival of the ingested bacteria inside the HKM inducing significant HKM cytotoxicity. The immunosuppressive effect of arsenic was further evident from the ability of A. hydrophila to colonize and disseminate efficiently in exposed fish. Enzyme linked immunosorbent assay indicated that chronic exposure to arsenic suppressed the production of pro-inflammatory 'IL-1beta like' factors from HKM. It is concluded that arsenic even at very low concentration is immunotoxic to fish and the changes observed in HKM may provide a useful early biomarker of low-level xenobiotic exposure.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
An Iranian soldier died at a toxicological intensive care unit at Munich seven days after a vesicant exposure. At the autopsy the typical symptoms of mustard gas intoxication were found. The vesicant was detected qualitatively by gas chromatography-mass spectrometry (GC-MS) in the abdominal fat and quantified in the tissues and in the body fluids by the following method: (1) extraction by dichloromethane, (2) cleanup of the extracts by thin-layer chromatography (TLC) on silica plates, (3) extractive derivatization with gold-chloride, and (4) quantitative determination by electrothermal atomic absorption spectrometry (ET-AAS). The equal extracts, after heating, served for blanks. The following concentrations were found (milligrams of mustard gas/kilograms of tissue wet weight): brain 10.7, cerebrospinal fluid 1.9, liver 2.4, kidney 5.6; spleen 1.5, lung 0.8, muscle 3.9, fat 15.1, skin 8.4, skin with subcutaneous fatty tissue 11.8, liquid from a skin blister: below detection limit, blood 1.1, and urine: below detection limit.
The purification of homogeneous glutathione S transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2 dichloro 4 nitrobenzene. Although each of the 4 enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p nitrobenzyl chloride. Assay conditions for a variety of substrates are included. All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.
Adult channel catfish were exposed to 0 or 15 ppm arsenic as sodium arsenate in synthetic lake water for approximately six months. After sacrifice, livers were removed and examined by conventional transmission electron microscopy, optical microscopy, scanning electron microscopy, and electron probe X-ray microanalysis. Cytoplasmic structures in the nuclei were found to contain highly concentrated deposits of iron. Thirty percent of these structures were also found to contain arsenic at a level above the minimum detectable level for the conditions of the study. The size, shape and relative position of the cytoplasmic structures were the same as the hemosiderin granules observed by conventional transmission electron microscopy. (JMT)
In 1973, the Special Studies Section of the Texas Water Quality Board first reported high levels of arsenic in Finfeather and Municipal lakes in Bryan, Texas in Brazos County (CEARLEY, unpublished). One source of the arsenic contamination was an industrial firm manufacturing arsenical dessicants used as cotton defoliants and arsenical pesticides. Seepage of arseniccontaminated wastes occurred from unreinforced waste treatment ponds into the headwaters of Finfeather Lake, approximately i km upstream from Municipal Lake. Levels of arsenic in the water were reported at 240 ppm in the headwaters of Finfeather Lake and at 0.30 ppm in Carters Creek, which was approximately 7 km from the manufacturing firm. These levels exceed the Texas Water Quality Board permissable discharge level of 0.05 ppm and the U. S. Public Health Service recommended drinking water level which is set at 0.01 ppm. Arsenic levels in sediments at a depth of 5 to 15 cm at I0 locations in Finfeather Lake ranged from i00 to 12,000 ppm and averaged 4700 ppm (CEARLEY, unpublished). Some tissues and the carcasses of seven fish species were observed to have accumulated arsenic to levels exceeding 0.5 ppm (dry weight) which is the Food and Drug Administration maximum permissable limit for arsenic in food. The liver is the target organ in arsenic poisoning of mammals (HARVEY 1975) and fish (SORENSEN et al. 1979a). Levels of arsenic in the livers of endemic populations of Lepomis cyanellus (green sunfish) and L. macrocharis (bluegills) averaged 2.2 ppm (+ 1.8 standard deviation). These levels were four orders of magnitude
Micronucleus assays with fish have been shown to be useful in vivo techniques for genotoxicity testing, and show potential for in situ monitoring of water quality. In this paper, we review the literature on the clastogenic effects of chemical and physical agents on fish cells, with emphasis on the induction of micronuclei in teleosts. Included in the review is a description of the mechanisms for formation of micronuclei in cells, and a summary of the various techniques that have been used for micronucleus analysis in fish. This review is directed to assisting laboratories in the development of fish genotoxicity assays for water quality monitoring.
Polychlorinated biphenyls (PCBs) are stable pollutants, which can be found in almost every compartment of terrestrial and aquatic ecosystems. They are very lipophilic and therefore have the potency of accumulating in the fat stores of animals. The mechanisms by which PCBs exert their adverse effects are still unclear. It is known that PCBs induce some important biotransformation enzymes, but their mutagenic properties are still controversial. The DNA breakage and clastogenic potency of a planar PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined in vivo in fish, using the single cell gel electrophoresis or comet assay and the micronucleus test, on erythrocytes of the brown trout exposed for 3, 9 and 14 days to initial PCB concentrations of 780 and 918 pg/ml, dissolved in the water. Blood was taken by a caudal puncture and the erythrocytes were either deposited in an agarose gel (0.6%) for the comet assay or smeared directly on slides for the micronucleus test. Five fish were studied per treatment and 50 and 2000 erythrocytes per concentration and per animal were analysed for the comet assay and the micronucleus test respectively. Ethyl methanesulphonate (EMS) at a concentration of 25 mg/l water was used as a positive control. Although EMS induced a statistically significant increase of single strand breaks in the comet assay, in neither of the two tests used, were mutagenic effects due to PCB exposure observed.
Mustard gas, bis(2-chloroethyl)sulfide, treatment of proteins is shown to generate significant amounts of covalently crosslinked protein dimers. This is due to the preferential alkylation of cysteine residues. Crosslinking does not occur in the model protein staphylococcal nuclease, which has no cysteine residues. Treatment of cysteine-containing mutants of staphylococcal nuclease with this chemical warfare agent did result in crosslinking. However, these dimers are slowly cleaved back to monomers by an unknown mechanism. The alkylation and crosslinking of cysteine-containing proteins by mustard gas may contribute to its toxicity.
Development, peak and healing lesions were induced in the skin of rabbits by topical applications (on different days) of the chemical irritant sulfur mustard (SM). Immediately after the rabbits were euthanized, the intact lesions were excised and organ-cultured for 17 to 20 hours. The culture fluids from early, peak and healing SM lesions all showed high chemotactic activity for both PMN and MN. This finding suggests that the PMN and MN, seen microscopically in tissue sections of the lesions, were entering continuously, even during the healing process. The chemotaxins identified were the eicosanoid LTB4, the chemokine IL-8, and proteases producing the complement fragment C5a. Other studies from our laboratory showed that the number of cells containing IL-1, IL-8, MCP-1, and GRO mRNAs was increased in SM lesions. Chemotactic activity was released by both live and dead (frozen and thawed) cell suspensions of PMN, MN, and fibroblasts, suggesting that these cells were major sources of the chemotaxins produced by the SM lesion explants. Explants of normal skin produced considerable chemotactic activity for MN, but not for PMN. Chemotactic activity for PMN, and the release of LTB4, IL-8 and proteases cleaving C5 to C5a, occurred only in explants infiltrated by leukocytes.
The study revealed that topically applied sulphur mustard, which is a potent blistering agent with mutagenic and carcinogenic properties, is also hepatotoxic. It produces severe steatosis and other pathological alterations, accompanied by biochemical changes. There is a significant rise in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvate transaminase (GPT) after exposure. The liver injury appeared to peak on the third day. Recovery at the ultrastructural level could be noticed on the sixth day but was far from complete because the gross pathological changes were still severe.
The article reports on the carcinogenicity bioassay of rainbow trout to determine the role of arsenic in fish carcinogenesis through a microinjection technique. The embryos of the trout were admitted in the laboratory at 10-11 degrees Celsius for about one week until they are hatched completely. In hatchery boxes, they were presented with 4-day long microinjection period wherein the temperature was maintained at 7-8 degreed Celsius to promote slow development. Results show that the incidence of the kidney lesions were present in fishes treated with arsenic and some were evident in liver of 4 specimens.
The ethoxyresorufin-O-deethylase (EROD) assay has been extensively used in whole animals and in cell culture as a biomarker of exposure to environmental contaminants such as dioxin-like compounds (DLCs). This paper addresses two controversial phenomena that arise when DLCs are examined by the EROD assay. Firstly, the maximum level of induced EROD activity varies with the identity of the inducing compound; secondly, the induced EROD activity reaches a concentration-dependent maximum level that is followed by an apparent reduction in activity when the concentration of inducer is further increased. These phenomena are completely explained by competitive inhibition of the EROD enzyme-substrate reaction by the dioxin-like compound. A kinetic model explains the biphasic appearance of EROD induction curves as a function of a compound's binding affinity with the Ah receptor (Kd) and its binding affinity to CYP 1A1 (Ki) which results in inhibition of the EROD enzyme-substrate reaction. These results limit the reliability of the information obtained from calibration curves of EROD activity versus concentration of a standard DLC such as 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Skin ulcers on fish are one of the most well-recognized indicators of polluted or otherwise stressed aquatic environments. In recent years, skin ulcer epidemics have been either experimentally or epidemiologically linked to exposure to a number of xenobiotic chemicals as well as to biotoxins. Some of these agents, such as toxins produced by the dinoflagellate alga Pfiesteria, have led to serious concerns about the health of aquatic ecosystems, such as estuaries along the east coast of the United States. However, a number of other risk factors besides Pfiesteria have been shown to damage epithelium and may also play important roles in skin ulcer pathogenesis. In addition, increasing evidence indicates that not only may skin damage occur via direct contact with toxins, but it may also be induced indirectly from physiological changes that result from exposure not only to toxins but also to other environmental stressors, such as pH and temperature extremes. The multifactorial pathways that operate at both the ecological and the organismal levels as well as the nonspecific response of the skin to insults make it very challenging to link epidemic skin ulcers to any single cause in natural aquatic populations. Consequently, using pathology to unequivocally identify the specific cause of a lesion (eg. Pfiesteria exposure) is not a valid approach. Only with an increased understanding of the basic mechanisms leading to skin damage (including development of specific biomarkers for specific toxins), along with a better understanding of ecological processes operating in these environments, will we be able to discern the relative importance of various risk factors in skin ulcer development.
Expression of cytochromes P450 (CYP) and glutathione S-transferases in the lung may be affected by inhaled pollutants. We have investigated the effect of sulfur mustard on the expression of CYP 1A1, 2B1, 2E1 and 3A1, as well as of alpha-, micro- and pi-glutathione S-transferases in rat lung. Sulfur mustard (0.025, 0.05 or 0.1 mg/kg) or its vehicle was administered to anaesthetized animals by intratracheal injection. Expression of CYP and glutathione S-transferases was analysed 24 hr after administration of the vesicant warfare using western blotting. Preservation of airway epithelium integrity after animal exposure to sulfur mustard was confirmed by histological examination of tracheal and lung tissues from control and treated animals. Constitutive levels of CYP 2B1 and 3A1 proteins were found in lung tissue from control rats, whereas CYP 1A1 and 2E1 proteins were not detected. Animal exposure to sulfur mustard enhanced CYP 3A1 protein levels by 80 to 103%. In contrast, exposure to sulfur mustard neither modified CYP 2B1 expression, nor led to detectable expression of CYP 1A1 or 2E1. Constitutive levels of alpha-, micro- and pi-glutathione S-transferase proteins were found in lung tissue from control rats. Exposure to sulfur mustard had no effect on expression of either of the glutathione S-transferases. Our results show that intratracheal exposure to sulfur mustard selectively increases CYP 3A1 expression in rat lung. Taking into account the major role of CYP of the 3A family in the metabolism of drugs, up-regulation of CYP 3A1 by sulfur mustard might have important therapeutic consequences.
Histopathological indicators and clinical observations were used to evaluate wound severity, depth and degree of healing on days 2 and 8 for full-skin-thickness sulfur-mustard (HD)-induced burns in weanling swine. Six female weanling swine were exposed for 2 h to 400 microl of HD at each of six dose sites on the hairless abdominal skin. Biopsy samples (8 mm) were taken from the periphery and from the center of the wound on day 2, and the wound was excised on day 8. Histopathological indicators evaluated were epidermal necrosis, follicular necrosis, dermal necrosis, vascular necrosis, depth of injury, ulceration (loss of epidermis), granulation tissue response, neovascularization, re-epithelialization (hyperplasia) and completeness of healing. Wounds were more severe from anterior to posterior. Histopathological assessment of epidermal ulceration and necrosis of epidermis, dermis, basal epithelium, adnexal structures and subcutaneous tissue were useful indicators of wound development on day 2. Granulation tissue response (observed as early as day 8) and re-epithelialization were good indicators of wound healing. Clinical evaluations were performed on day 2 prior to and after debriding, and on study day 8. Clinical observations on study day 2 were for wound size and for exudation, erythema, edema, necrosis and eschar. Clinical observations on study day 8 were for the previous parameters and for re-epithelialization, granulation and infection. Wound size and severity increased from anterior to posterior position. Size, exudation and edema were useful indicators of wound development. These histological and clinical observation parameters will be used in future experiments to compare various treatments for HD-induced burns.