http://informahealthcare.com/cot ISSN: 1556-9527 (print), 1556-9535 (electronic) Cutan Ocul Toxicol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15569527.2014.975241
RESEARCH ARTICLE
Acute and long-term ocular effects of acrolein vapor on the eyes and potential therapies
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Shlomit Dachir1, Maayan Cohen1, Hila Gutman1, Liat Cohen1, Hillel Buch1, and Tamar Kadar1 Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel
Abstract
Keywords
Objective: Acrolein is a potent irritant and a vesicant that was used by the French during WWI as the warfare agent named: ‘‘papite’’. Nowadays, it is produced in large amounts all over the world in the industry for the production of acrylic acid and for agriculture use as herbicide. The aim of this study was to characterize the effects of acute eye exposure to acrolein vapor and to evaluate the efficacy of a topical post-exposure combination treatment with a local anesthetic and a steroid. Methods: Rabbit eyes were exposed to three doses of acrolein vapor (low, intermediate and high) and treated topically with either 0.4% benoxinate hydrochloride (localin, for 2 h) or dexamethasone (dexamycin, for 6 days) or a combination of both drugs. Clinical follow-up using slit lamp examinations and histological evaluation was performed 4 weeks post-exposure. Results: Acrolein vapor caused immediate eye closure with excess tearing, corneal erosions and severe inflammation of the anterior chamber. This was followed by corneal neovascularization (NV) starting as early as 1 week post-exposure. The damage to the eyes was long lasting, and at 4 weeks following exposure, significant pathological changes were observed. Immediate postexposure application of the local anesthetic, localin, prevented the eye closure, and the dexamycin treatment reduced significantly the initial inflammation as well as the extent and incidence of corneal NV. Conclusions: Short-term eye exposure to the irritant acrolein may result in immediate eye closure and long lasting pathologies that could lead to blindness. An anti-inflammatory treatment combined with short-term application of a local anesthetic prevents incapacitation and might minimize significantly the extent of eye injuries.
Acrolein, eyes, irritant, localin, steroids
Introduction The hazard of exposure to toxic industrial chemicals (TICs) such as acrolein had become a realistic threat. Many TICs are routinely used in industry, agriculture and research, and the current first aid treatments include only immediate rinsing with water. Moreover, no specific antidote exists and the treatment typically consists of removing the chemical substance from the body (skin and or eyes) as soon as possible and providing supportive medical care in a hospital setting as needed (www.cdc.org). Acrolein is well known for its pungent odor and its strong irritating effects on mucous membranes, especially the eyes and upper respiratory tract1. The main source of everyday exposure to this irritant is from atmospheric acrolein that is released from incomplete organic combustion such as residential fireplaces, burning coal, oil and gas, automobile exhaust, overheated vegetable and animal fats and tobacco
Address for correspondence: Shlomit Dachir, Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, 74100 Israel. E-mail: [email protected]
History Received 27 July 2014 Revised 18 September 2014 Accepted 3 October 2014 Published online 30 October 2014
smoke causing slow oxidative damage to various body organs2–4. Yet, acrolein is also produced commercially world-wide since 1938, and it is used mainly as an herbicide to control aquatic plants and alga in irrigation canals and water recirculating systems2,3. It is also used as an intermediate product in many manufacturing processes, especially in the production of acrylic acid. In 1991, it was estimated that over 113 ton/year were produced worldwide2. According to the EPA5 reports from 2008, about 500 ton of acrolein are sold annually in the USA only to use as herbicides. Therefore, there is a risk of accidental exposure to this chemical. Moreover, because of its lachrymatory and vesicant properties, it was used by the French during World War I (WWI) as a warfare agent ‘‘papite’’2. At present, no specific treatment is known to prevent its deleterious effects. Acrolein can cause eye irritation in a concentration as low as 0.1–0.2 mg/m3. A concentration of 0.34 mg/m3 results in nose irritation; however, its odor can be identified only at a concentration of about 0.48 mg/m3 in the air. The biological effects of acrolein are thought to be a consequence of its reactivity toward nucleophiles such as
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cysteine, lysine, histidine and arginine residues in critical regions of various proteins6,7. It is an a,b-unsaturated aldehyde that contains a reactive carbonyl group and an electrophilic a-carbon3. It alkylates proteins and nucleic acids within the cells and inhibits synthesis of proteins, RNA as well as DNA2,3,8. Upon entering the body, acrolein is captured by glutathione resulting in a dose-dependent significant decrease in glutathione levels, a decrease that might have deleterious toxicological effects. Depletion of glutathione pool limits the ability of the cells to cope with various reactive metabolites. Moreover, acrolein inhibits detoxification of exogenous substances by binding to cytochrome p-450 in the liver3,9,10. Most of the research that was conducted to study the effects of exposure to acrolein deals with its effects on the respiratory system in general and on the upper parts in particular. The majority of these studies relate to cases of chronic exposure to low doses2,11. To the best of our knowledge, no experimentally controlled research was performed to investigate the effects of acute ocular exposure to acrolein vapor. Therefore, this study is aimed to characterize the effects of acute eye exposure to acrolein vapor using the rabbit eye model which is a well-established model for studying chemical injuries and test potential therapy to avoid the severe damage to the eyes12–14.
Materials and Methods
Cutan Ocul Toxicol, Early Online: 1–8
different levels of injuries in order to characterize its dosedependent effects on the eyes. Post-exposure treatment with dexamycin (dexamethasone sodium phosphate 1 mg/ml and neomycin sulfate 5 mg/ml, Teva, Israel) Dexamycin is an anti-inflammatory aimed to reduce the development of inflammation following exposure to acrolein vapor (intermediate and high dose). 50 ml of dexamycin drops were applied to the eyes four times a day, for 6 days, starting 1 h following exposure. Efficacy of a combined treatment with dexamycin and localin (benoxinate hydrochloride 0.4%, Fischer) (a) Localin is a local anesthetic aimed to reduce pain. Rabbits were exposed to the high dose of acrolein vapor and were then divided into three groups: (1) control non-treated, (2) treated with 50 ml saline droplets, every 15 min, starting 10 min post-exposure for the duration of 2 h (total of eight treatments). (3) Treated with 50 ml localin droplets, every 15 min, starting 10 min post-exposure for the duration of 2 h (total of eight treatments). (b) Localin and dexamycin were applied following exposure to acrolein vapor (intermediate and high dose) with the same frequency and dosing as for the single treatments.
Animals
Clinical evaluation
New-Zealand white female rabbits (Harlan, Israel), weighing 2–3 kg were housed in individual mesh cages in the animal facilities. Rooms were temperature controlled (21 ± 1 C), with lights on from 06:00 to 18:00, and rabbits had ad libitum access to food (Altromin 2023 GmbH, Germany) and water. All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, 1996, and were approved by the Institutional Animal Care and Use Committee of IIBR.
Clinical follow-up. Clinical follow-up was conducted daily
Exposure procedure Rabbit eyes were exposed to different concentrations of acrolein vapor (90%, Sigma-Aldrich, Israel) aiming to produce severe, moderate and mild injuries. Acrolein (10, 20 or 30 ml) was applied to a filter paper disc held within a glass goggle that was attached to the rabbit eye for a period of 4 min. The liquid evaporated within the goggle producing concentrations of 0.25 up to 0.8 mg/ml (these are the highest calculated values possible, assuming the goggles were completely sealed). The whole procedure was performed in a hood. Experimental outline Characterization of acrolein induced eye lesions Rabbits’ eyes were exposed to different doses of acrolein vapor (low, intermediate and high dose) resulting in three
during the first 5 days following exposure and once a week there-after until the end of the experiment. Detailed slit-lamp examinations of each eye were carried out. Corneal epithelial defects were observed by fluorescein staining (Bio Glo, fluorescein sodium ophthalmic strips, HUB Pharmaceuticals, LLC, Rancho Cucamonga, CA). The clinical findings were documented by photography and scored according to our clinical severity scoring scale15. Corneal neovascularization (NV) was scored as peripheral or central depending on the length of the vessels. The area of the erosions was calculated from digital photographs, using morphometric analysis with an image analysis system (Image-Pro Express 4.0).
Morphometric analysis of erosions area.
Corneal thickness, as a parameter for corneal edema, was measured at the center of the cornea with an ultrasonic pachymeter (SP-3000, Tomey). Before pachymetry, eyes were locally anesthetized using topical 0.4% Benoxinate HCL (localin ophthalmic solution).
Corneal thickness.
Histology Following euthanasia, rabbit eyes were enucleated and fixed in 4% neutral-buffered paraformaldehyde (NBPF) and further processed routinely for paraffin embedding.
DOI: 10.3109/15569527.2014.975241
Representative 5 mm paraffin sections were stained with Hematoxylin & Eosin (H&E) for general morphology. Statistics Results are expressed as mean ± standard error of the mean (s.e.m.). The quantitative data were subjected to statistical analysis using one-way and two-way ANOVA utilizing SPSS software (version 14.0, SPSS Inc., Chicago, IL). Posthoc comparisons were performed using the simple main effects contrasts analysis for interactions and Bonferoni test for multiple comparisons.
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Results Clinical observations Exposure to acrolein induced, in some of the rabbits, eye irritation and rhinorrhea already during the exposure procedure itself. As early as 10 min after exposure, most of the rabbits closed their eyes (Figure 1). Five hours Corneal erosions and inflammation of the anterior segment were observed shortly after exposure including swelling of the eyelids and conjunctiva (Figure 1). The extent of inflammation was dose dependent, exhibiting extremely swollen eyelids mainly in the group that was exposed to the highest dose of acrolein. It was not clear what the extent of erosions was at this time point (5 h) in those eyes due to their almost complete closure. Following exposure to the intermediate dose of acrolein, only superficial erosions were observed and only in part of the eyes.
Eye exposure to vapors of the irritant acrolein
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Twenty four hours Following exposure, the acute damage to the eyes was maximal. Severe inflammation of the anterior chamber including substantial swelling of the eyelids was still observed in the rabbits exposed to the higher dose of acrolein. Tear secretion was increased in all exposed rabbits, even in those with relatively slight injuries. Edema that was demonstrated by opacity developed in more than 50% of the corneas exposed to the higher dose while in the eyes exposed to the lower dose, this phenomenon was not observed. Erosions appeared in the corneas of 60, 75 and 100% of the eyes that were exposed to low, intermediate and high doses, respectively. In this time point, of 24 h post-exposure, it was still difficult to perform clinical examination to some of the eyes that were exposed to the highest dose, due to the severe inflammation and edema of the eyelids. One to four weeks The clinical findings up to 4 weeks post-exposure are presented in Figure 2. Most of the clinical signs of inflammation disappeared within 1–2 weeks, yet, in the eyes exposed to the highest dose no apparent reduction of the inflammation and edema was observed. As early as 1 week following exposure, a process of NV in some of the eyes commenced. The in-growth of blood vessels into the cornea was found mainly in those eyes that were exposed to the highest concentration of acrolein and that exhibited very severe acute injuries. No NV growth was observed in the eyes that were exposed to the lowest dose of acrolein and in the intermediate concentration only superficial NV developed in a small number of eyes and most of them disappeared with time.
Figure 1. Five hours following exposure to acrolein vapor. A complete eye closure is observed, including extensive tear secretion (A intermediate dose). Note the dose–response effect and the severe edema in the conjunctiva and eyelids (B low dose versus C high dose).
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Cutan Ocul Toxicol, Early Online: 1–8
Figure 2. Quantitative evaluation of the acute and late clinical findings up to 4 weeks following exposure to various concentrations of acrolein vapor (A). The clinical severity score of the corneas includes in-growth of blood vessels (NV) into the cornea. (B) An example of corneal NV 4 weeks following exposure to a high dose of acrolein vapor. The clinical score of the high-dose group was significantly more severe than that of the two lower doses (p50.001–0.05). Significant differences were also demonstrated from week 1 up to 3 weeks between the two lower doses groups (low versus intermediate dose, p50.05, n ¼ 8–10 eyes in each group).
Figure 3. Representative photographs of corneal erosions 24 h following 4 min exposure to low (A) and high (B) doses of acrolein vapor. The graph of the erosions area (C) demonstrates the significant dose–response between the damage to the corneal epithelium and the dose of acrolein vapor (two way ANOVA, p50.001 high dose versus intermediate and lower doses, n ¼ 8–10 eyes in each group).
Morphometric analysis Morphometric analysis revealed correlation between erosions area and the dose of acrolein exposure: the largest erosions were found in the eyes exposed to the highest doses
(Figure 3B and C). Erosions area reached a peak at 24 h following exposure. Eyes with moderate exposures had smaller erosion area (Figure 3A) that healed within 48 h (low dose) or 96 h (intermediate dose). The large erosions that developed following exposure to the highest dose healed only 1 week following exposure (Figure 3C).
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DOI: 10.3109/15569527.2014.975241
Eye exposure to vapors of the irritant acrolein
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Figure 4. Histological micrographs representing naı¨ve cornea (A) and corneas that were exposed to high concentration of acrolein vapor (B–D) 4 weeks following exposure. Note the pathological findings in the exposed corneas: thin epithelium, inflammatory cells, NV (B), goblet cells (arrows in C) and damaged endothelium (D). H&E staining, objective magnification 20 (A, B, D), 40 (C).
Histological examination
Effect of localin on eye-closure
Histological examination revealed significant changes 4 weeks following exposure to the highest concentration of acrolein (Figure 4). The epithelial layer was thin compared to normal epithelium, and the stroma was edematous exhibiting infiltration of inflammatory cells (Figure 4B and C). Migration of conjunctival goblet cells was observed in the epithelial surface of some of the corneas (Figure 4C) indicating the development of limbal stem cell deficiency (LSCD). Moreover, in some of the eyes, the endothelium, the most inner layer of the cornea, was also damaged (Figure 4D). Yet, in eyes that were exposed to the intermediate concentration, only slight changes were observed in the corneal epithelium.
The efficacy of localin application on preventing photophobia and eye closure was tested following exposure to the highest dose of acrolein. These rabbits closed their eyes immediately following exposure and the treatment with localin enabled them to open the eyes. Eyes that were treated with saline exhibited complete eye closure similar to the untreated group. About 2 h following exposure, at the completion of the treatment with localin, half to completely open eyes were observed in the treated rabbits, while in untreated and saline-treated rabbits, the eyes were closed at this point. At 24 h post-exposure, all rabbits were scored 0.5, a score that expressed partial closure of the eyes, independent of the initial treatment they received. The aim of the treatment with localin was to reduce pain and therefore prevent eye closure; yet, follow-up of the acute damage in the anterior chamber up to 1 week postexposure showed a significant advantage (p50.024) to the treatment with localin demonstrating a decrease in the clinical score compared to saline treatment or no treatment at all (Figure 6). The repeated rinsing of the eyes with saline during 2 h following exposure did not result in opening of the eyes or in any reduction in the extent of the damage compared to the non-treated eyes. Moreover, in the eyes treated with localin, the corneal thickness (Figure 6B) was reduced during the first 24 h following exposure to acrolein, and the extent of NV development was substantially reduced up to 4 weeks (data not shown).
Post-exposure treatment with dexamycin The post-exposure treatment with the steroid dexamycin (DEX) was very effective in reducing the extent of the damage (Figure 5). A significant decrease in the acute ocular lesion was found (p50.009 treated versus acrolein non-treated animals) and in the extent of the edema that was demonstrated by a decrease in the corneal thickness (Figure 5A). The reduction in the clinical severity score of the late pathology in the cornea, including neovascularization, was statistically significant only in those rabbits that were exposed to the high dose of acrolein vapor (Figure 5B).
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Cutan Ocul Toxicol, Early Online: 1–8
Figure 5. The effect of DEX treatment on corneal thickness (A) and on the acute and delayed pathology (B) up to 4 weeks following exposure to two doses of acrolein vapor: high and intermediate. DEX significantly ameliorated corneal edema in both experimental groups (A; p50.009 acrolein (high and intermediate dose) versus acrolein + DEX) as assessed by corneal thickness. Late pathology was reduced by DEX only in the high-dose group p50.001–0.05 acrolein (high and intermediate, n ¼ 12 eyes in each group) versus acrolein + DEX.
Figure 6. The effect of localin on the extent of acute corneal damage (A) and on corneal thickness (B) following exposure to a high dose of acrolein (n ¼ 12 in each group). Note the significant decrease in the clinical severity score following treatment with localin compared to saline alone (two way ANOVA, p50.024) (A). Corneal thickness was reduced during the first 24-h post-exposure (B) as assessed by corneal thickness.
Efficacy of a combined treatment with dexamycin and localin After demonstrating the efficacy of localin and dexamycin, separately, the efficacy of a combined treatment with both drugs was tested. Application of localin drops during the first 2 h after exposure and treatment with dexamycin, starting 1 h after exposure for the duration of 6 days. As expected, the combined treatment decreased significantly the acute damage, reduced the late pathology and decreased corneal thickness as measured by pachimetry (Figure 7), along with preventing eye closure following exposure.
Discussion Acrolein is an extremely volatile aldehyde, a strong irritant and a vesicant that was used by the French as a warfare agent during WWI. Today, acrolein is commercially produced in large quantities, which makes it highly available. Most of the knowledge about the risks posed by acrolein deal with
inhalation exposure and injury to the respiratory system following incidents of acute or chronic exposure to very low doses. This study focused on the evaluation of the extent of acrolein effects in a scenario of acute eye exposure to different vapor concentrations and on the efficacy of treatment with local anesthetic and steroid. Acrolein is well known to cause severe eye irritation accompanied by pain and tearing following exposure. Minor irritation might appear following exposure to only 0.5 ppm. Its high solubility in water might be one of the reasons for its severe effects on mucus membranes such as the cornea3. In general, the extent of ocular injury is dependent on the duration of exposure and on the concentration of acrolein in the air1,16. In the current study, similar to evidence in the literature, acrolein was found to be extremely irritant. In the vapor concentrations tested, even in the low dose, rabbits closed their eyes and exhibited significant tearing within a few minutes of exposure. Later, erosions were observed and severe inflammation including edema developed in the cornea and in a more prominent way in the
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DOI: 10.3109/15569527.2014.975241
Eye exposure to vapors of the irritant acrolein
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Figure 7. The effect of a combined treatment with localin and dexamycin (DEX) on the corneal thickness (A) and on the acute and delayed pathology of the cornea (B) up to 4 weeks following exposure to two doses of acrolein vapor: high and medium doses (n ¼ 12 in each group). (B) p50.02 treated versus acrolein non-treated, (C) p50.05 treated versus acrolein non-treated.
conjunctiva. Following exposure to the low-acrolein concentrations clinical recovery was observed within 4–7 days; however, following exposure to the high dose, at 1 week no healing was observed and the cornea deteriorated. The reaction of acrolein with cysteine, histidine and lysine residues of proteins and with nucleophilic sites in DNA is probably the basis for the cytotoxicity in the cells exposed to high concentration of acrolein6,17. Acrolein is also capable of reacting with glutathione creating an oxidative stress effect on cells inhibiting healing processes4,18. Monitoring the eyes up to 4 weeks post-exposure revealed a long-term pathology and the development of limbal stem cells deficiency (LSCD) as was shown by histology, associated with NV. A similar pattern of long-term pathology was previously described by us in eyes following exposure to sulfur mustard (SM)15,19. The appearance of limbal damage associated with NV and conjunctivalization of the corneal epithelium is characteristic of LSCD. After demonstrating the immediate and severe eye injury following acute short-term exposure, a combined treatment starting with application of the local anesthetic localin followed by anti-inflammatory treatment with dexamycin was tested. Localin was used for relief enabling the rabbits to open their eyes. This treatment was previously found efficient in preventing incapacitation following eye exposure to sulfur mustard (SM) (data not published). Similarly, in the current study, localin proved to be very effective in preventing eye closure. Interestingly, following exposure to acrolein vapor, localin was not only anesthetic but it also reduced the extent of edema and of late pathology. This effect was not a result of the eye washing itself since a similar treatment with saline did not result in eye opening and did not reduce the late pathology. This improvement may be due to the localin mechanism of action as an anesthetic. Some of the anesthetic substances, such as localin, are calmodulin antagonists20,21. It is possible that increase in Ca+2 is involved in the development of the injury and therefore the use of localin might inhibit Ca+2-phospholipase A2 and the release of arachidonic acid which might contribute to the reduction in the extent of injury.
Further, a post-exposure treatment with the steroid dexamycin was investigated. This treatment was previously shown to reduce the acute inflammatory reaction following eye exposure to SM vapor and to delay appearance of late pathology. Moreover, a symptomatic treatment was found to decrease the NV growth into the cornea22. Similarly, in the current study a post-exposure treatment was very effective in reducing the extent of the acute as well as the late pathology after exposure to acrolein vapor. There was a significant decrease in the inflammation and edema of the cornea as well as the incidence of NV growth. In conclusion, it was shown that short-term exposure to acrolein vapor resulted in immediate eye closure and long lasting severe damage to the eyes which can be devastating. A short-term application of local anesthetic prevented eye closure, thus avoids incapacitation, and the treatment with the anti-inflammatory steroid decreased significantly the extent of the acute and late pathologies.
Declaration of interest The authors report no conflicts of interest.
References 1. Beauchamp Jr RO,, Andjelkovich DA, Kligerman AD, et al. A critical review of the literature on acrolein toxicity. Crit Rev Toxicol 1985;14:309–379. 2. Ghilarducci DP, Tjeerdema RS. Fate and effects of acrolein. Rev Environ Contam Toxciol 1995;144:95–146. 3. Bein K, Leikauf GD. Acrolein – a pulmonary hazard. Mol Nutr Food Res 2011;55:1342–1360. 4. Li L, Holian A. Acrolein: a respiratory toxin that suppresses pulmonary host defense. Rev Environ Health 1998;13:99–108. 5. US EPA. Reregistration eligibility decision: acrolein. Office of Prevention, Pesticides and Toxic Substances (7508P); 2008. Available from: www.epa.gov/oppsrrd1/. . ./acrolein_red 6. Aldini G, Orioli M, Carini M. Protein modification by acrolein: relevance to pathological conditions and inhibition by aldehyde sequestering agents. Mol Nutr Food Res 2011;55:1301–1319. 7. Stevens JF, Maier CS. Acrolein: sources, metabolism and biomolecular interactions relevant to human health and disease. Mol Nutr Food Res 2008;52:7–25.
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8. Li H, Want J, Kaphalia BS, Ansari GAS, Khan MF. Quantitation of acrolein-protein adducts: potential biomarker of acrolein exposure. J Toxicol Environ Health 2004;67:513–524. 9. Monteil C, Prieur EL, Buisson S, et al. Acrolein toxicity: comparative in vitro study with lung slices and pneumocytes type II cell line from rats. Toxicology 1999;133: 129–138. 10. Pizzimenti S, Ciamporcero E, Daga M, et al. Interaction of aldehydes derived from lipid peroxidation and membrane proteins. Front Physiol 2013;4:1–17. 11. Feron VJ, Kruysse A, Til HP, Immel HR. Repeated exposure to acrolein vapour: subacute studies in hamsters, rats and rabbits. Toxicology 1978;9:47–57. 12. Esquenazi S, Bazan HEP, Bui V, et al. Topical combination of NGF and DHA increases rabbit corneal nerve regeneration after photorefractive keratectomy. Invest Ophthalmol Vis Sci 2005;46: 3121–3127. 13. Kadar T, Turetz J, Fisbine E, et al. Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits. Curr Eye Res 2001;22:42–53. 14. Lallemand F, Furrer P, Felt-Baeyens O, et al. A novel watersoluble cyclosporine A prodrug: ocular tolerance and in vivo kinetics. Int J Pharm 2005;295:7–14.
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15. Kadar T, Dachir S, Cohen L, et al. Ocular injuries following sulfur mustard exposure – pathological mechanism and potential therapy. Toxicology 2009;263:59–69. 16. Darley EF, Middleton JT, Garber MJ. Plant damage and eye irritation from ozone-hydrocarbon reactions. J Agric Food Chem 1960;8:483–485. 17. Kehrer JP, Biswal SS. The molecular effects of acrolein. Toxicol Sci 2000;57:6–15. 18. Adams JD, Klaidman LK. Acrolein-induced oxygen radical formation. Free Radic Biol Med 1993;15:187–193. 19. Kadar T, Horwitz V, Sahar R, et al. Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits. Curr Eye Res 2011;36: 1098–1107. 20. Kadar T, Fishbeine E, Meshulam Y, et al. Treatment of skin injuries induced by sulfur mustard with calmodulin antagonists, using the pig model. J Appl Toxicol 2000;20:S133–S136. 21. Sawyer TW, Conley J, Hunter K, et al. Domestic swine model for the assessment of chemical warfare agent-anesthetic interactions: some effects of sulfur mustard. Mil Med 2000;165:573–578. 22. Amir A, Turetz J, Chapman S, et al. The beneficial effects of topical anti-inflammatory drugs against HD-induced ocular lesions in rabbits. J Appl Toxicol 2000;20:S109–S114.
RESEARCH ARTICLE
Acute and long-term ocular effects of acrolein vapor on the eyes and potential therapies
Cutaneous and Ocular Toxicology Downloaded from informahealthcare.com by Fudan University on 05/16/15 For personal use only.
Shlomit Dachir1, Maayan Cohen1, Hila Gutman1, Liat Cohen1, Hillel Buch1, and Tamar Kadar1 Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel
Abstract
Keywords
Objective: Acrolein is a potent irritant and a vesicant that was used by the French during WWI as the warfare agent named: ‘‘papite’’. Nowadays, it is produced in large amounts all over the world in the industry for the production of acrylic acid and for agriculture use as herbicide. The aim of this study was to characterize the effects of acute eye exposure to acrolein vapor and to evaluate the efficacy of a topical post-exposure combination treatment with a local anesthetic and a steroid. Methods: Rabbit eyes were exposed to three doses of acrolein vapor (low, intermediate and high) and treated topically with either 0.4% benoxinate hydrochloride (localin, for 2 h) or dexamethasone (dexamycin, for 6 days) or a combination of both drugs. Clinical follow-up using slit lamp examinations and histological evaluation was performed 4 weeks post-exposure. Results: Acrolein vapor caused immediate eye closure with excess tearing, corneal erosions and severe inflammation of the anterior chamber. This was followed by corneal neovascularization (NV) starting as early as 1 week post-exposure. The damage to the eyes was long lasting, and at 4 weeks following exposure, significant pathological changes were observed. Immediate postexposure application of the local anesthetic, localin, prevented the eye closure, and the dexamycin treatment reduced significantly the initial inflammation as well as the extent and incidence of corneal NV. Conclusions: Short-term eye exposure to the irritant acrolein may result in immediate eye closure and long lasting pathologies that could lead to blindness. An anti-inflammatory treatment combined with short-term application of a local anesthetic prevents incapacitation and might minimize significantly the extent of eye injuries.
Acrolein, eyes, irritant, localin, steroids
Introduction The hazard of exposure to toxic industrial chemicals (TICs) such as acrolein had become a realistic threat. Many TICs are routinely used in industry, agriculture and research, and the current first aid treatments include only immediate rinsing with water. Moreover, no specific antidote exists and the treatment typically consists of removing the chemical substance from the body (skin and or eyes) as soon as possible and providing supportive medical care in a hospital setting as needed (www.cdc.org). Acrolein is well known for its pungent odor and its strong irritating effects on mucous membranes, especially the eyes and upper respiratory tract1. The main source of everyday exposure to this irritant is from atmospheric acrolein that is released from incomplete organic combustion such as residential fireplaces, burning coal, oil and gas, automobile exhaust, overheated vegetable and animal fats and tobacco
Address for correspondence: Shlomit Dachir, Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, 74100 Israel. E-mail: [email protected]
History Received 27 July 2014 Revised 18 September 2014 Accepted 3 October 2014 Published online 30 October 2014
smoke causing slow oxidative damage to various body organs2–4. Yet, acrolein is also produced commercially world-wide since 1938, and it is used mainly as an herbicide to control aquatic plants and alga in irrigation canals and water recirculating systems2,3. It is also used as an intermediate product in many manufacturing processes, especially in the production of acrylic acid. In 1991, it was estimated that over 113 ton/year were produced worldwide2. According to the EPA5 reports from 2008, about 500 ton of acrolein are sold annually in the USA only to use as herbicides. Therefore, there is a risk of accidental exposure to this chemical. Moreover, because of its lachrymatory and vesicant properties, it was used by the French during World War I (WWI) as a warfare agent ‘‘papite’’2. At present, no specific treatment is known to prevent its deleterious effects. Acrolein can cause eye irritation in a concentration as low as 0.1–0.2 mg/m3. A concentration of 0.34 mg/m3 results in nose irritation; however, its odor can be identified only at a concentration of about 0.48 mg/m3 in the air. The biological effects of acrolein are thought to be a consequence of its reactivity toward nucleophiles such as
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cysteine, lysine, histidine and arginine residues in critical regions of various proteins6,7. It is an a,b-unsaturated aldehyde that contains a reactive carbonyl group and an electrophilic a-carbon3. It alkylates proteins and nucleic acids within the cells and inhibits synthesis of proteins, RNA as well as DNA2,3,8. Upon entering the body, acrolein is captured by glutathione resulting in a dose-dependent significant decrease in glutathione levels, a decrease that might have deleterious toxicological effects. Depletion of glutathione pool limits the ability of the cells to cope with various reactive metabolites. Moreover, acrolein inhibits detoxification of exogenous substances by binding to cytochrome p-450 in the liver3,9,10. Most of the research that was conducted to study the effects of exposure to acrolein deals with its effects on the respiratory system in general and on the upper parts in particular. The majority of these studies relate to cases of chronic exposure to low doses2,11. To the best of our knowledge, no experimentally controlled research was performed to investigate the effects of acute ocular exposure to acrolein vapor. Therefore, this study is aimed to characterize the effects of acute eye exposure to acrolein vapor using the rabbit eye model which is a well-established model for studying chemical injuries and test potential therapy to avoid the severe damage to the eyes12–14.
Materials and Methods
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different levels of injuries in order to characterize its dosedependent effects on the eyes. Post-exposure treatment with dexamycin (dexamethasone sodium phosphate 1 mg/ml and neomycin sulfate 5 mg/ml, Teva, Israel) Dexamycin is an anti-inflammatory aimed to reduce the development of inflammation following exposure to acrolein vapor (intermediate and high dose). 50 ml of dexamycin drops were applied to the eyes four times a day, for 6 days, starting 1 h following exposure. Efficacy of a combined treatment with dexamycin and localin (benoxinate hydrochloride 0.4%, Fischer) (a) Localin is a local anesthetic aimed to reduce pain. Rabbits were exposed to the high dose of acrolein vapor and were then divided into three groups: (1) control non-treated, (2) treated with 50 ml saline droplets, every 15 min, starting 10 min post-exposure for the duration of 2 h (total of eight treatments). (3) Treated with 50 ml localin droplets, every 15 min, starting 10 min post-exposure for the duration of 2 h (total of eight treatments). (b) Localin and dexamycin were applied following exposure to acrolein vapor (intermediate and high dose) with the same frequency and dosing as for the single treatments.
Animals
Clinical evaluation
New-Zealand white female rabbits (Harlan, Israel), weighing 2–3 kg were housed in individual mesh cages in the animal facilities. Rooms were temperature controlled (21 ± 1 C), with lights on from 06:00 to 18:00, and rabbits had ad libitum access to food (Altromin 2023 GmbH, Germany) and water. All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, 1996, and were approved by the Institutional Animal Care and Use Committee of IIBR.
Clinical follow-up. Clinical follow-up was conducted daily
Exposure procedure Rabbit eyes were exposed to different concentrations of acrolein vapor (90%, Sigma-Aldrich, Israel) aiming to produce severe, moderate and mild injuries. Acrolein (10, 20 or 30 ml) was applied to a filter paper disc held within a glass goggle that was attached to the rabbit eye for a period of 4 min. The liquid evaporated within the goggle producing concentrations of 0.25 up to 0.8 mg/ml (these are the highest calculated values possible, assuming the goggles were completely sealed). The whole procedure was performed in a hood. Experimental outline Characterization of acrolein induced eye lesions Rabbits’ eyes were exposed to different doses of acrolein vapor (low, intermediate and high dose) resulting in three
during the first 5 days following exposure and once a week there-after until the end of the experiment. Detailed slit-lamp examinations of each eye were carried out. Corneal epithelial defects were observed by fluorescein staining (Bio Glo, fluorescein sodium ophthalmic strips, HUB Pharmaceuticals, LLC, Rancho Cucamonga, CA). The clinical findings were documented by photography and scored according to our clinical severity scoring scale15. Corneal neovascularization (NV) was scored as peripheral or central depending on the length of the vessels. The area of the erosions was calculated from digital photographs, using morphometric analysis with an image analysis system (Image-Pro Express 4.0).
Morphometric analysis of erosions area.
Corneal thickness, as a parameter for corneal edema, was measured at the center of the cornea with an ultrasonic pachymeter (SP-3000, Tomey). Before pachymetry, eyes were locally anesthetized using topical 0.4% Benoxinate HCL (localin ophthalmic solution).
Corneal thickness.
Histology Following euthanasia, rabbit eyes were enucleated and fixed in 4% neutral-buffered paraformaldehyde (NBPF) and further processed routinely for paraffin embedding.
DOI: 10.3109/15569527.2014.975241
Representative 5 mm paraffin sections were stained with Hematoxylin & Eosin (H&E) for general morphology. Statistics Results are expressed as mean ± standard error of the mean (s.e.m.). The quantitative data were subjected to statistical analysis using one-way and two-way ANOVA utilizing SPSS software (version 14.0, SPSS Inc., Chicago, IL). Posthoc comparisons were performed using the simple main effects contrasts analysis for interactions and Bonferoni test for multiple comparisons.
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Results Clinical observations Exposure to acrolein induced, in some of the rabbits, eye irritation and rhinorrhea already during the exposure procedure itself. As early as 10 min after exposure, most of the rabbits closed their eyes (Figure 1). Five hours Corneal erosions and inflammation of the anterior segment were observed shortly after exposure including swelling of the eyelids and conjunctiva (Figure 1). The extent of inflammation was dose dependent, exhibiting extremely swollen eyelids mainly in the group that was exposed to the highest dose of acrolein. It was not clear what the extent of erosions was at this time point (5 h) in those eyes due to their almost complete closure. Following exposure to the intermediate dose of acrolein, only superficial erosions were observed and only in part of the eyes.
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Twenty four hours Following exposure, the acute damage to the eyes was maximal. Severe inflammation of the anterior chamber including substantial swelling of the eyelids was still observed in the rabbits exposed to the higher dose of acrolein. Tear secretion was increased in all exposed rabbits, even in those with relatively slight injuries. Edema that was demonstrated by opacity developed in more than 50% of the corneas exposed to the higher dose while in the eyes exposed to the lower dose, this phenomenon was not observed. Erosions appeared in the corneas of 60, 75 and 100% of the eyes that were exposed to low, intermediate and high doses, respectively. In this time point, of 24 h post-exposure, it was still difficult to perform clinical examination to some of the eyes that were exposed to the highest dose, due to the severe inflammation and edema of the eyelids. One to four weeks The clinical findings up to 4 weeks post-exposure are presented in Figure 2. Most of the clinical signs of inflammation disappeared within 1–2 weeks, yet, in the eyes exposed to the highest dose no apparent reduction of the inflammation and edema was observed. As early as 1 week following exposure, a process of NV in some of the eyes commenced. The in-growth of blood vessels into the cornea was found mainly in those eyes that were exposed to the highest concentration of acrolein and that exhibited very severe acute injuries. No NV growth was observed in the eyes that were exposed to the lowest dose of acrolein and in the intermediate concentration only superficial NV developed in a small number of eyes and most of them disappeared with time.
Figure 1. Five hours following exposure to acrolein vapor. A complete eye closure is observed, including extensive tear secretion (A intermediate dose). Note the dose–response effect and the severe edema in the conjunctiva and eyelids (B low dose versus C high dose).
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Figure 2. Quantitative evaluation of the acute and late clinical findings up to 4 weeks following exposure to various concentrations of acrolein vapor (A). The clinical severity score of the corneas includes in-growth of blood vessels (NV) into the cornea. (B) An example of corneal NV 4 weeks following exposure to a high dose of acrolein vapor. The clinical score of the high-dose group was significantly more severe than that of the two lower doses (p50.001–0.05). Significant differences were also demonstrated from week 1 up to 3 weeks between the two lower doses groups (low versus intermediate dose, p50.05, n ¼ 8–10 eyes in each group).
Figure 3. Representative photographs of corneal erosions 24 h following 4 min exposure to low (A) and high (B) doses of acrolein vapor. The graph of the erosions area (C) demonstrates the significant dose–response between the damage to the corneal epithelium and the dose of acrolein vapor (two way ANOVA, p50.001 high dose versus intermediate and lower doses, n ¼ 8–10 eyes in each group).
Morphometric analysis Morphometric analysis revealed correlation between erosions area and the dose of acrolein exposure: the largest erosions were found in the eyes exposed to the highest doses
(Figure 3B and C). Erosions area reached a peak at 24 h following exposure. Eyes with moderate exposures had smaller erosion area (Figure 3A) that healed within 48 h (low dose) or 96 h (intermediate dose). The large erosions that developed following exposure to the highest dose healed only 1 week following exposure (Figure 3C).
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DOI: 10.3109/15569527.2014.975241
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Figure 4. Histological micrographs representing naı¨ve cornea (A) and corneas that were exposed to high concentration of acrolein vapor (B–D) 4 weeks following exposure. Note the pathological findings in the exposed corneas: thin epithelium, inflammatory cells, NV (B), goblet cells (arrows in C) and damaged endothelium (D). H&E staining, objective magnification 20 (A, B, D), 40 (C).
Histological examination
Effect of localin on eye-closure
Histological examination revealed significant changes 4 weeks following exposure to the highest concentration of acrolein (Figure 4). The epithelial layer was thin compared to normal epithelium, and the stroma was edematous exhibiting infiltration of inflammatory cells (Figure 4B and C). Migration of conjunctival goblet cells was observed in the epithelial surface of some of the corneas (Figure 4C) indicating the development of limbal stem cell deficiency (LSCD). Moreover, in some of the eyes, the endothelium, the most inner layer of the cornea, was also damaged (Figure 4D). Yet, in eyes that were exposed to the intermediate concentration, only slight changes were observed in the corneal epithelium.
The efficacy of localin application on preventing photophobia and eye closure was tested following exposure to the highest dose of acrolein. These rabbits closed their eyes immediately following exposure and the treatment with localin enabled them to open the eyes. Eyes that were treated with saline exhibited complete eye closure similar to the untreated group. About 2 h following exposure, at the completion of the treatment with localin, half to completely open eyes were observed in the treated rabbits, while in untreated and saline-treated rabbits, the eyes were closed at this point. At 24 h post-exposure, all rabbits were scored 0.5, a score that expressed partial closure of the eyes, independent of the initial treatment they received. The aim of the treatment with localin was to reduce pain and therefore prevent eye closure; yet, follow-up of the acute damage in the anterior chamber up to 1 week postexposure showed a significant advantage (p50.024) to the treatment with localin demonstrating a decrease in the clinical score compared to saline treatment or no treatment at all (Figure 6). The repeated rinsing of the eyes with saline during 2 h following exposure did not result in opening of the eyes or in any reduction in the extent of the damage compared to the non-treated eyes. Moreover, in the eyes treated with localin, the corneal thickness (Figure 6B) was reduced during the first 24 h following exposure to acrolein, and the extent of NV development was substantially reduced up to 4 weeks (data not shown).
Post-exposure treatment with dexamycin The post-exposure treatment with the steroid dexamycin (DEX) was very effective in reducing the extent of the damage (Figure 5). A significant decrease in the acute ocular lesion was found (p50.009 treated versus acrolein non-treated animals) and in the extent of the edema that was demonstrated by a decrease in the corneal thickness (Figure 5A). The reduction in the clinical severity score of the late pathology in the cornea, including neovascularization, was statistically significant only in those rabbits that were exposed to the high dose of acrolein vapor (Figure 5B).
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Figure 5. The effect of DEX treatment on corneal thickness (A) and on the acute and delayed pathology (B) up to 4 weeks following exposure to two doses of acrolein vapor: high and intermediate. DEX significantly ameliorated corneal edema in both experimental groups (A; p50.009 acrolein (high and intermediate dose) versus acrolein + DEX) as assessed by corneal thickness. Late pathology was reduced by DEX only in the high-dose group p50.001–0.05 acrolein (high and intermediate, n ¼ 12 eyes in each group) versus acrolein + DEX.
Figure 6. The effect of localin on the extent of acute corneal damage (A) and on corneal thickness (B) following exposure to a high dose of acrolein (n ¼ 12 in each group). Note the significant decrease in the clinical severity score following treatment with localin compared to saline alone (two way ANOVA, p50.024) (A). Corneal thickness was reduced during the first 24-h post-exposure (B) as assessed by corneal thickness.
Efficacy of a combined treatment with dexamycin and localin After demonstrating the efficacy of localin and dexamycin, separately, the efficacy of a combined treatment with both drugs was tested. Application of localin drops during the first 2 h after exposure and treatment with dexamycin, starting 1 h after exposure for the duration of 6 days. As expected, the combined treatment decreased significantly the acute damage, reduced the late pathology and decreased corneal thickness as measured by pachimetry (Figure 7), along with preventing eye closure following exposure.
Discussion Acrolein is an extremely volatile aldehyde, a strong irritant and a vesicant that was used by the French as a warfare agent during WWI. Today, acrolein is commercially produced in large quantities, which makes it highly available. Most of the knowledge about the risks posed by acrolein deal with
inhalation exposure and injury to the respiratory system following incidents of acute or chronic exposure to very low doses. This study focused on the evaluation of the extent of acrolein effects in a scenario of acute eye exposure to different vapor concentrations and on the efficacy of treatment with local anesthetic and steroid. Acrolein is well known to cause severe eye irritation accompanied by pain and tearing following exposure. Minor irritation might appear following exposure to only 0.5 ppm. Its high solubility in water might be one of the reasons for its severe effects on mucus membranes such as the cornea3. In general, the extent of ocular injury is dependent on the duration of exposure and on the concentration of acrolein in the air1,16. In the current study, similar to evidence in the literature, acrolein was found to be extremely irritant. In the vapor concentrations tested, even in the low dose, rabbits closed their eyes and exhibited significant tearing within a few minutes of exposure. Later, erosions were observed and severe inflammation including edema developed in the cornea and in a more prominent way in the
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DOI: 10.3109/15569527.2014.975241
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Figure 7. The effect of a combined treatment with localin and dexamycin (DEX) on the corneal thickness (A) and on the acute and delayed pathology of the cornea (B) up to 4 weeks following exposure to two doses of acrolein vapor: high and medium doses (n ¼ 12 in each group). (B) p50.02 treated versus acrolein non-treated, (C) p50.05 treated versus acrolein non-treated.
conjunctiva. Following exposure to the low-acrolein concentrations clinical recovery was observed within 4–7 days; however, following exposure to the high dose, at 1 week no healing was observed and the cornea deteriorated. The reaction of acrolein with cysteine, histidine and lysine residues of proteins and with nucleophilic sites in DNA is probably the basis for the cytotoxicity in the cells exposed to high concentration of acrolein6,17. Acrolein is also capable of reacting with glutathione creating an oxidative stress effect on cells inhibiting healing processes4,18. Monitoring the eyes up to 4 weeks post-exposure revealed a long-term pathology and the development of limbal stem cells deficiency (LSCD) as was shown by histology, associated with NV. A similar pattern of long-term pathology was previously described by us in eyes following exposure to sulfur mustard (SM)15,19. The appearance of limbal damage associated with NV and conjunctivalization of the corneal epithelium is characteristic of LSCD. After demonstrating the immediate and severe eye injury following acute short-term exposure, a combined treatment starting with application of the local anesthetic localin followed by anti-inflammatory treatment with dexamycin was tested. Localin was used for relief enabling the rabbits to open their eyes. This treatment was previously found efficient in preventing incapacitation following eye exposure to sulfur mustard (SM) (data not published). Similarly, in the current study, localin proved to be very effective in preventing eye closure. Interestingly, following exposure to acrolein vapor, localin was not only anesthetic but it also reduced the extent of edema and of late pathology. This effect was not a result of the eye washing itself since a similar treatment with saline did not result in eye opening and did not reduce the late pathology. This improvement may be due to the localin mechanism of action as an anesthetic. Some of the anesthetic substances, such as localin, are calmodulin antagonists20,21. It is possible that increase in Ca+2 is involved in the development of the injury and therefore the use of localin might inhibit Ca+2-phospholipase A2 and the release of arachidonic acid which might contribute to the reduction in the extent of injury.
Further, a post-exposure treatment with the steroid dexamycin was investigated. This treatment was previously shown to reduce the acute inflammatory reaction following eye exposure to SM vapor and to delay appearance of late pathology. Moreover, a symptomatic treatment was found to decrease the NV growth into the cornea22. Similarly, in the current study a post-exposure treatment was very effective in reducing the extent of the acute as well as the late pathology after exposure to acrolein vapor. There was a significant decrease in the inflammation and edema of the cornea as well as the incidence of NV growth. In conclusion, it was shown that short-term exposure to acrolein vapor resulted in immediate eye closure and long lasting severe damage to the eyes which can be devastating. A short-term application of local anesthetic prevented eye closure, thus avoids incapacitation, and the treatment with the anti-inflammatory steroid decreased significantly the extent of the acute and late pathologies.
Declaration of interest The authors report no conflicts of interest.
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