Sulphur mustard induced DNA damage in mice after dermal and inhalation exposure

Toxicology 139 (1999) 39 – 51 www.elsevier.com/locate/toxicol

Sulphur mustard induced DNA damage in mice after dermal and inhalation exposure P.V. Lakshmana Rao *, R. Vijayaraghavan, A.S.B. Bhaskar Di6ision of Pharmacology and Toxicology, Defence Research and De6elopment Establishment, Jhansi Road, Gwalior 474002, India Received 29 April 1999; accepted 28 July 1999

Abstract Sulphur mustard (SM) is a chemical warfare agent of the blistering agent category for which there is still no effective therapy. SM, being a strong electrophile, readily reacts with a wide range of cellular macromolecules including DNA, RNA and protein. Since the main intoxication routes for SM are inhalation and dermal penetration, in the present study we have exposed female mice to different concentrations of SM by dermal and inhalation exposures and estimated the DNA damage in different organs viz., liver, lung, spleen and thymus. SM was applied at 38.7, 77.4, 154.7 mg/kg body weight, on the hair-clipped skin (dermal exposure) equivalent to 0.25, 0.5 and 1.0 of the LD50. Inhalation exposure was carried out at 10.6, 21.2 and 42.3 mg/m3 for 1 h duration equivalent to 0.25, 0.5 and 1.0 LC50. SM induced a dose-dependent DNA damage in all the organs except the lung in dermal exposure. Similarly the inhalation exposure resulted in dose- and time-dependent effect in all the organs including lung. By both routes of exposure liver was the most affected organ followed by spleen, thymus and lung in decreasing order. The quantitative data were corroborated by qualitative analysis of DNA on agarose gel electrophoresis. The genomic DNA analysis of the organs had revealed random nuclear DNA fragmentation resulting in a ‘smear’ typical of necrotic form of cell death. Since DNA damage is not reversible especially in liver, this can be used as a marker for SM exposure through either the dermal or inhalation route. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemical warfare agent; Bis (2-chloroethyl) sulphide; Dermal; DNA damage; Inhalation; Sulphur mustard

1. Introduction

Abbre6iations: DAPI, 4%,6-diamidino-2-phenylindole; GSH, glutathione; NAD+, nicotinamide adenine dinucleotide; PTFE, polytetrafluoroethylene; SM, sulphur mustard. * Corresponding author. Fax: +91-751-341148. E-mail address: [email protected] (P.V. Lakshmana Rao)

Sulphur mustard (SM), commonly known as mustard gas (bis [2-chloroethyl] sulphide), is an alkylating agent and causes blisters upon contact with human skin. SM is a frequently used chemical warfare agent and several reports are available of its recent use (Somani and Babu, 1989). The eyes, the skin and the respiratory tract are the principal target organs of SM toxicity (Papirmeis-

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ter et al., 1991; Pechura and Rall, 1993). SM is highly lipophilic and is absorbed very quickly through the skin. After a latent period of 6 – 24 h erythema and blisters appear on the skin. Generally in the animal’s skin blisters do not appear but otherwise, the lesions are similar to those observed in humans. Pulmonary complications, mainly on the upper respiratory tract, such as haemorrhagic inflammation, sore throat, hoarseness, cough, bronchitis and bronchopneumonia are observed in SM-exposed victims (Freitag et al., 1991). Additionally lung cancers has been reported in fishermen exposed to SM and in workers of SM manufacturing plants (Iwaszkiewicz, 1966; Aasted et al., 1987; Easton et al., 1988). The mechanism of SM-induced injury is not fully understood and no effective antidote is known. There is no effective method for evaluating the efficacy of therapeutic agents in preventing SM-induced injury to human tissues. A number of mechanisms have been proposed for the cytotoxicity of SM including DNA damage, labilization of lysosomes and calcium mediated toxicity. DNA appears to be a critical target and alkylation of nucleic acids is an early molecular event for SM induced toxicity (Papirmeister et al., 1985). Exposure to a low level of SM has been demonstrated to result in depression of DNA synthesis in bacteria, and mammalian cells (Papirmeister et al., 1985; Somani and Babu, 1989; Ribeiro et al., 1991). There are only very few studies following inhalation exposure to SM (Calvet et al., 1994; Vijayaraghavan, 1997). The effects of inhaled SM on DNA damage in various organs in animal models are not available in the literature (Pechura and Rall, 1993). Since the main routes of entry of SM are dermal or by inhalation, in the present study we have exposed mice to different doses of SM by skin application and by inhalation route. The objective of the present study was to estimate the DNA damage qualitatively and quantitatively in various vital organs viz., lung, liver, spleen and thymus and time, dose- and route-dependent changes for an assessment of this parameter as a potential biological marker for SM exposure.

2. Materials and methods

2.1. Chemicals SM was synthesised in the Synthetic Chemistry Division of our Establishment and was found to be more than 99% pure by gas chromatographic analysis. All other chemicals used were of extra pure grade and obtained from Sigma Chemical Co. (St Louis, MO, USA) and E. Merck, India Ltd. (Bombay).

2.2. Animals Swiss albino female mice weighing between 24 and 26 g body weight from the Establishment’s animal facility were used for the study. The animals were housed in polypropylene cages with dust-free rice husk as the bedding material, and were provided with pellet food (Amrut Laboratory Feeds, Maharashtra, India) and water ad libitum. Animals were exposed to SM either dermally or by inhalation. This study has the approval of the Institute’s Ethical Committee.

2.3. Dermal exposure SM was diluted freshly in polyethylene glycol 300 and was applied uniformly, using a micro syringe on to the back of the mice on a circular area of 1.5 cm diameter, after closely clipping the hair (the hair was clipped 24 h before the application). The dilutions were such that not more than 100 ml was applied. The SM was applied as a single dose of 38.7, 77.4 and 154.7 mg/kg body weight equivalent to 0.25, 0.5 and 1.0 LD50, respectively (Vijayaraghavan et al., 1991). The body weight of animals were recorded daily, and possible mortalities were counted.

2.4. Inhalation exposure The inhalation exposure chamber consisted of a 50 cm long and 10 cm diameter cylindrical chamber made of polytetrafluoroethylene (PTFE), positioned horizontally for exposing 10 mice at a time, exposure being of head only. The mice were exposed in individual body plethysmographs made

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of glass. The generation of SM vapours and the analysis are given in detail elsewhere (Vijayaraghavan, 1997). Briefly, SM was diluted in 10 ml of acetone and the solution was vaporised in a compressed air nebuliser. The vapours were directed into the exposure chamber, and the outgoing air from the exposure chamber was decontaminated and then exhausted out in a fume hood (Fig. 1). The chamber air was analysed gas chromatographically. The mice were exposed only once to various concentrations of SM vapours at 10.6, 21.2, and 42.3 mg/m3 for 1 h (equivalent to 0.25, 0.5 and 1.0 LC50, respectively). During inhalation exposure, four mice per concentration were monitored for respiratory modification. A differential pressure transducer was attached to the body plethysmograph for the measurement of respiratory flow. The flow signals from the individual transducers were amplified using an universal amplifier (Gould, Cincinnati, USA). The amplified signals were digitised using an analog to digital converter (Metrabyte, Taunton, USA), integrated as tidal volume (VT),

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stored and analysed using a personal computer. A computer program capable of recognising the effects of airborne chemicals as sensory irritation, airway obstruction and pulmonary irritation, and a combination of these effects, described earlier was used (Vijayaraghavan et al., 1994). All the respiratory variables viz., respiratory frequency (f), VT, inspiratory time (TI) and expiratory time (TE) were also measured using the computer program.

2.5. Experimental protocol Three doses per dermal route and three concentrations per inhalation route were used. The body weight of the animals was recorded and four animals from each group were sacrificed at 1, 2 and 7 days post-exposure. Animals which survived for 14 days were also sacrificed and samples were collected. The animals were sacrificed by cervical dislocation and lung, liver, spleen and thymus were quickly removed, washed free of blood and frozen at −20°C before assay.

Fig. 1. Schematic diagram of sulphur mustard (SM) exposure assembly. The exposure chamber is made of polytetrafluoroethylene (PTFE) with detachable glass body plethysmograph for head only exposure of mice to SM vapours. Differential pressure transducer-critical orifice assembly is used for sensing respiratory flow.

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2.6. DNA fragmentation assay

3. Results

DNA fragmentation was assayed as previously described (Rao et al., 1998). Briefly, the organs viz. liver, lung, spleen and thymus from control and treated animals were quickly excised and frozen. The frozen tissues were homogenised in ice-cold lysis buffer (10 mM Tris, 20 mM EDTA, 0.5% Triton X-100, pH 8.0) and then centrifuged at 27 000× g for 30 min. Both pellet (intact chromatin) and supernatant (DNA fragments) were assayed for DNA content fluorimetrically by using fluorescent dye 4%,6-diamidino-2-phenylindole (DAPI) (Brunk et al., 1979). Briefly, to 2 ml of the reagent 100 ng/ml DAPI in 10 mM Tris (pH 7.4) containing 100 mM NaCl, 20 ml of a sample were added and then fluorescence intensity was measured at 450 nm with excitation at 362 nm. The percentage of fragmented DNA was defined as the ratio of the DNA content of the supernatant at 27 000×g to the total DNA in the lysate (Wyllie, 1980).

A progressive dose-dependent fall in body weights of animals was observed in both the routes of exposure. Decrease in body weights started after 2 days of exposure (data not shown). SM-exposed animals showed less feed and water intake than the control animals. Although mice did not show any blister formation, lesions started appearing after 1 week on the site of dermal exposure. Since the inhalation exposure was head only exposure, the heads of the animals were swollen and the eyes were closed because of edema of lids. The animals were less active. SMexposed animals by both the routes showed significant decrease in the weight of liver, spleen and thymus with duration and dose. Thymus was obliterated in dermal exposure after 7 days.

2.7. DNA agarose gel electrophoresis Genomic DNA from the four organs (lung, liver, spleen and thymus) of control and treated animals was extracted (Prigent et al., 1993) and damage was assessed qualitatively by agarose gel electrophoresis using 1.2% agarose in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0). Hind III, and Hind III/EcoR1 double digests of l phage DNA served as molecular size standard. The gels were visualised after ethidium bromide staining.

2.8. Statistical analysis The data were analysed by two way analysis of variance and Student’s-Newman-Keul’s multiple comparisons procedure was used for finding the difference between the various exposed groups. A probability of P 5 0.05 was considered statistically significant. SigmaStat (Jandel Scientific Corporation, CA, USA) was used for statistical analysis.

3.1. Dermal exposure DNA damage at different doses and time intervals was estimated as percent DNA fragments in lung, liver, spleen and thymus (Fig. 2). The percent DNA fragments in control animals varied from 10.9 to 13.7 and there was no appreciable variation between different organs. However, in SM treated animals there was dose and time dependent increase in DNA fragments in all the organs except lung. No DNA damage was observed either quantitatively or qualitatively at any of the doses and time durations in the lungs. Percent DNA fragments increased with duration recording the maximum damage (52.89 3.5%) 7 days post-exposure at 154.7 mg/kg dose. Spleen and thymus also showed similar patterns of DNA damage. There was no significant increase in DNA damage in the thymus with dose, at 7 days post-exposure. Agarose gel electrophoresis of genomic DNA isolated from different organs of the control mice showed a single high molecular weight band indicating the integrity of DNA (Fig. 3). In confirmation of the quantitative data on DNA fragmentation, the qualitative analysis also did not show any damage in lung. A single high molecular weight band (Fig. 4a–c lanes 1, 5 and 9) can be seen, whereas genomic DNA of liver, spleen and thymus of treated animals showed

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exposure is summarised in Fig. 8. The DNA damage after inhalation exposure also showed dose and time-dependent effects. DNA fragmentation in four organs of the control group varied from 11.0 to 13.0%. At a concentration of 10.6 mg/m3 SM, there was no significant difference in DNA damage in liver, spleen and thymus at 1, 2, 7 days post-exposure. At 10.6 mg/ m3, there was moderate increase in DNA fragmentation in lung tissues until 48 h post-exposure but subsequently recovered to control values by 7th day. However, at concentrations of 21.2 and 42.3 mg/m3, the DNA damage increased with the duration of exposure. The maximum damage of 36.8% DNA fragmentation in lung was observed at 42.3 mg/m3 concentration and the damage persisted until 7th day. There was progressive increase in DNA damage in liver, spleen and thymus with increasing concentrations and duration of exposure. The liver was the most affected organ followed by the spleen, thymus and lung in decreasing order. Fig. 2. Percent DNA fragments in liver, lung, spleen and thymus of control and SM treated percutaneous application of sulphur mustard (SM) at 38.7, 77.4 and 154.7 mg/kg body weight of animals at different time intervals. Values are mean9SE of four animals each. *Means followed by the same alphabet(s) are not significantly different at P B0.05 by Dunnet’s multiple comparison test.

varying degrees of necrotic damage seen as a smear. (Fig. 4a–c). The damage was more pronounced with increasing dose in liver and spleen. The quantitative data is well corroborated by qualitative analysis.

3.2. Inhalation exposure During exposure to SM the mice showed sensory irritation, 15–20 min after the start of exposure, characterised by a pause between the inspiration and the expiration (Fig. 5). The respiratory frequency decreased progressively reaching a stable state. Other respiratory variables were also changed as expected for a sensory irritant particularly the time of brake (Figs. 6 and 7). After stopping SM exposure, recovery of the respiratory rate was observed. The quantitative estimate of percent DNA fragments after inhalation

Fig. 3. Agarose gel electrophoresis of genomic DNA isolated from liver, lung, and spleen of control animals. Key: M-l phage DNA Hind III digest; 1- lung; 2- liver; 3- spleen; 4thymus.

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Agarose gel electrophoresis of genomic DNA from four organs of control animals did not show any damage (figure not shown). As compared to the controls, different organs showed a varying degree of damage (Fig. 9a–c). At 10.6 mg/m3 SM concentration, lung showed mild DNA damage at 24 and 48 h but by 7th day presence of single band indicates that repair processes were under way. At 21.2 and 42.3 mg/m3 the damage was moderate but persistent. The DNA damage profile of liver, spleen, and thymus is similar to that observed after dermal exposure of SM.

4. Discussion Several hypotheses have been proposed to explain SM-induced toxicity. According to Papirmeister et al., (1985) production of DNA breaks by mustard activates the chromosomal

Fig. 4. Agarose gel electrophoresis of genomic DNA isolated from liver, lung, spleen and thymus of animals after dermal application of different doses of SM. (a) 38.7 mg/kg [0.25 LD50]; (b) 77.4 mg/kg [0.50 LD50]; (c) 154.7 mg/kg [1.0 LD50]. Key: M- l phage DNA Hind III / EcoR1 double digest; 1–4: 24 h; 5 – 8: 48 h; 9 – 12: 7 days post-exposure of lung, liver, spleen, and thymus, respectively.

Fig. 5. Typical recording of respiratory flow obtained during exposure to sulphur mustard vapours. (a) control, (b) during exposure to 10.6 mg/m3 (c) control and (d) during exposure to 42.3 mg/m3.

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duced DNA damage and various adducts formed, there are very limited reports on the dose- and time-dependent effect of SM induced DNA damage in various vital organs especially after inhalation exposure. The present study was carried out to evaluate SM-induced DNA damage in four major organs liver, lung, spleen and thymus after dermal and inhalation exposures. The primary target organ for the airborne chemicals is the lung, because of its close proximity to the environment (Poynter and Ballantyne, 1977). Low dose inhalation exposure of mustard gas causes respiratory-depression; more intense exposure causes sneezing, lacrimation, rhinorrhea, nasal bleeding, burning throat, hoarseness and hacking cough (Somani and Babu, 1989). In the present study, exposure to SM vapours induced

Fig. 6. Time response analysis for breath classification and measured variables for a group of four mice: before, during and after exposure to 10.6 mg m − 3 of sulphur mustard vapours. Sigma Plot 2.0 for windows is used for plotting the graph. The arrow indicates the duration of exposure. N, normal; S, sensory irritation; A, airflow limitation; P, pulmonary irritation; and SA, SP, PA and SPA are combinations of breath classification. VT, tidal volume; TI, time of inspiration; TE, time of expiration; VD, mid expiratory flow; TB, time of brake; TP, time of pause; and f, respiratory frequency.

enzyme poly (ADP ribose) polymerase, which in turn depletes cellular nicotinamide adenine dinucleotide (NAD+), inhibits glycolysis, and ultimately causes cell death. Another mechanism suggested for SM induced cytotoxicity is based on lipid peroxidation occurring as a result of the formation of reactive oxygen intermediates as a consequence of glutathione (GSH) depletion (Papirmeister et al., 1991; Vijayaraghavan et al., 1991). Depletion of GSH can lead to increase in intracellular Ca2 + leading to cell death (Orrenius et al.,, 1989). Although numerous reports have provided data on the initial reaction of SM in-

Fig. 7. Time response analysis for breath classification and measured variables for a group of four mice: before, during and after exposure to 42.3 mg/m3 (1.0 LC50) of sulphur mustard vapours. Sigma Plot 2.0 for windows is used for plotting the graph. The arrow indicates the duration of exposure. For abbreviations see Fig. 3.

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Fig. 8. Percent DNA fragments in liver, lung, spleen and thymus of control and SM after inhalation exposure at animals at different time intervals. Values are mean 9 SE of four animals each. *Means followed by the same alphabet(s) are not significantly different at PB0.05 by Dunnet’s multiple comparison test.

sensory irritation and the effect was delayed. Chemicals that stimulate trigeminal or laryngeal nerve endings show a typical sensory irritation pattern. The respiratory frequency decreases because of the sensory irritation (Alarie, 1966). The sensory irritation was reversible at lower vapour concentrations showing that it may be a direct effect of SM on the sensory nerve endings. However, inhalation of SM in mice has been shown to induce airway obstruction on subsequent days (Vijayaraghavan, 1997). Intratracheal infusion of SM in guinea pigs caused an increase in respiratory resistance and microvascular permeability which may be responsible for the airway obstruction (Calvet et al., 1994). A decrease in the body weight of the animals especially after percutaneous application is consistent with other reports. No mortality was observed until 5th day by either route of exposure.

Fig. 9. Agarose gel electrophoresis of genomic DNA isolated from liver, lung, spleen and thymus of animals after inhalation exposure of different concentrations of SM. (a) 10.6 mg/m3 (0.25 LC50); (b) 21.2 mg/m3 (0.5 LC50); (c) 42.3 mg/m3 (1.0 LC50). Key: M- l phage DNA Hind III /EcoR1 double digest; 1 – 4: 24 h; 5 – 8: 48 h; 9 – 12: 7 days post-exposure of lung, liver, spleen, and thymus, respectively.

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Mortality was observed after 7th day at 77.4 and 154.7 mg/kg and none of the animals survived by the 10th day post-exposure and only one animal survived up to the 14th day at 38.7 mg/kg dose. In the case of inhalation exposure there was no mortality until 14 days at 10.6 and 21.2 mg/m3 dose but 75.0% mortality was observed after 10th day at 42.3 mg/m3. A considerable decrease in size of spleen and thymus was observed. At higher concentrations of SM by both the routes there was obliteration of thymus. Similar observation was reported in another study where a single dermal application of sublethal doses (15.5, 7.75, and 3.88 mg/kg) of SM in Balb C mice resulted in a dose-dependent decrease in body weight, organ body weight ratio and cellularity of spleen and thymus after 7 days (Venkateswaran et al., 1994). The most important molecular target of SM is DNA because this agent alkylates and cross-links the purine bases (Fox and Scott, 1980). The alkylation of DNA by mustards has been reported by several workers (Lawley and Brooks, 1965; Ludlum et al., 1984). The DNA cross-links in C3H10 T1/2 cells have been investigated by Murnane and Byfield (1981) who found that bifunctional mustards increase the DNA cross-links by several fold leading to cell death or malfunctioning. In the present study, we have observed a definite doseand time-dependent DNA damage after percutaneous and inhalation exposures. Except for the lack of DNA damage in lung tissue after percutaneous application, all the other organs viz., liver, spleen and thymus showed necrotic DNA damage. Persistence of DNA damage in liver, spleen and thymus even after 7 days post-exposure is an important observation in the present study. This could be partly be a result of the cascade of events triggered by free-radical generation and partly to mismatch in DNA repair process. Ribeiro et al. (1991) employed nuclear sedimentation assay for the determination of DNA damage in cultures exposed to primary cultures of rat cutaneous keratinocytes. Their results showed that within 1 h of exposure to 0.1 mM SM structural integrity of cellular DNA was compromised and reduced the repair process even after 72 h post-exposure. Following cutaneous application of

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SM which reflects one of the most likely routes of poisoning in man, it was established that 93– 95% of the applied dose was absorbed within 6 h, and of this 60–75% was excreted within 8 days in the urine. However, the prolonged elimination of hydrolysis products caused by slow turn over/ breakdown of proteins and nucleic acids (Hambrook et al., 1992) has been reported. Such a phenomenon suggests indirectly persistence of DNA breakage long after the exposure to SM. Although the lungs have been reported to be the major target organ for SM-induced injury, in our study no DNA damage was observed after dermal application of SM. Agarose gel electrophoresis of lung DNA also did not show any damage as only a single high molecular weight band was seen. However, DNA damage was observed in lung after inhalation exposure. There are no reports with animal studies on the effects of SM in lung tissue after percutaneous exposure. When [14C] SM was injected intravenously (10 mg/kg), a significant amount of [14C] was detected within hours in kidney, liver, lung, intestine and stomach, organs all associated with the elimination and transformation processes (Maisonneuve et al., 1994). One interesting observation in our study is a moderate to severe DNA damage in liver by both the routes of SM exposure. There was dose-dependent damage which was not repaired with time. Earlier we reported depletion of GSH and hepatic lipid peroxidation after dermal application of SM at 38.7 and 77.4 mg/kg (Vijayaraghavan et al., 1991). The effects on DNA have been generally related to the genotoxic properties of SM but not much importance is paid to its possible contribution to hepatotoxic potential. It is not currently known whether SM-induced DNA damage plays any role in hepatic necrosis. Many of the hepatotoxins (carbon tetrachloride, cyclophosphamide, aflatoxin B1, etc.) that both alkylate cellular macromolecules and produce acute necrosis also alkylate DNA and initiate chronic responses including neoplasia. The analgesic acetaminophen (paracetamol) is a known hepato-

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toxicant that directly alkylates genomic DNA in vivo and in vitro and produces genotoxic effects in varied systems (Dybing et al., 1984). Dabrowska et al., (1996) have shown SM-induced apoptosis and necrosis, in endothelial cells in vitro. Rikimaru et al., (1991) have shown that when organ cultures of full-thickness human skin explants were exposed to high concentrations of SM, it caused rapid necrosis thereby preventing any active response from the injured cells. Both necrosis and apoptosis produce changes in common, including nuclear Ca2 + deregulation and DNA cleavage. There is other evidence that apoptosis and necrosis have select molecular changes in common. Although we anticipated low molecular weight DNA fragments or apoptotic mode of DNA damage (DNA ‘ladder pattern’) in spleen and thymus because of decrease in their size with duration, no such pattern was observed. One possible explanation could be the concentration of SM used in the present study may be higher and the time interval selected may be inadequate to detect such an event. It is well accepted that apoptosis and necrosis are part of a cell death continuum. Mild damage or early events of severe damage tend to trigger apoptosis because required signalling networks are intact and ATP is available to implement energy-dependent death programmes. With a more severe insult or at later stages of damage necrosis becomes the more favoured mode of cell death (Dabrowska et al., 1996). The DNA damage observed at all the SM concentrations used in the present study may be a result of severe toxic insult. Further studies are required to delineate the role of DNA damage in the possible hepatotoxic potential of SM as liver is a major target organ of SM induced toxicity either after a single low dose or repeated doses. In conclusion, the present study indicates that dermal or inhalation exposure of SM can lead to persistent DNA damage in vital organs such as liver, lung, spleen and thymus and can also contribute to systemic toxicity by other biochemical pathways. Since DNA damage is not reversible particularly in the liver this can be used as a biomarker for the alleged SM exposure either by dermal or inhalation route.

Acknowledgements The authors are grateful to Dr K. Sugendran for critical suggestions and Dr R.V.Swamy, Director, Defence Research and Development Establishment for encouragement and support.

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Further Reading Koschier, F.J., 1999. Toxicity of middle distillates from dermal exposure. Drug Chem. Toxicol. 22 (1), 155 – 164. Abstract This report focuses on recent studies that investigated the effects of kerosine dermal exposure on neurotoxicity and reproductive/developmental toxicity. Background toxicity information will also be reviewed for kerosine range mid distillates. The kerosine range mid distillates have a carbon range of C9 – C16 and have a boiling range of 302 – 554°F (150 – 290°C). This category includes kerosine, aviation fuels (e.g. Jet A, JP-5, and JP-8), no. 1 fuel oil and diesel fuel oil. In general, the kerosine range mid distillates demonstrate relatively low acute toxicity by any route of exposure. High inhalation exposures can induce central nervous system depression characterized by ataxia, hypoactivity, and prostration. Kerosines are known to cause skin irritation and inflammation under conditions of acute and repeated exposure in animals and humans, but are only slightly irritating to the eye and are not skin sensitizers. In addition, the absorption of kerosine range mid distillates through the skin has been demonstrated to be fairly rapid, but limited to approximately 10 – 15% of the applied dose after 24 h. The kerosine range mid distillates are generally inactive in genetic toxicity tests although positive studies have been reported. Positive results, while at times equivocal, have been reported for straight run kerosine and jet fuel A in the mouse lymphoma assay with metabolic activation, and hydrodesulfurized kerosine (mouse) and jet fuel A (rat) in the bone marrow cytogenetic assay. Effects on the nervous and reproductive systems have been reported in humans and experimental animals under conditions where inhalation and dermal exposure to specific kerosine type fuels are sometimes difficult to separate. Recent laboratory studies have addressed this point and examined the effects of dermal exposure. In these studies, rats were exposed to hydrodesulfurized kerosine by skin application to determine the potential of dermal contact to cause reproductive/developmental toxicity (OECD Guideline 421) or neurotoxicity (TSCA Guidelines on subchronic inhalation and neurotoxicity studies). These studies demonstrated that the highest dose level of kerosine does not induce reproductive/developmental or neurotoxicity effects by skin exposure in rodent studies. The dermal NOEL for HDS kerosine in rats was o`494 mg/kg for both neurotoxicity, and reproductive/developmental toxicity. Loizou, G.D., Jones, K., Akrill, P., Dyne, D., Cocker, J., 1999. Estimation of the dermal absorption of m-xylene vapor in humans using breath sampling and physiologi-

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cally based pharmacokinetic analysis. Toxicol. Sci. 48 (2), 170 – 179. Abstract A physiologically-based pharmacokinetic model, containing a skin compartment, was derived and used to simulate experimentally-determined exposure to m-xylene, using human volunteers exposed under controlled conditions. Biological monitoring was conducted by sampling, in exhaled alveolar air and blood, mxylene and urinary methyl hippuric acid concentrations. The dermal absorption of m-xylene vapor was successfully and conveniently studied using a breath sampling technique, and the contribution to m-xylene body burden from the dermal route of exposure was estimated to be 1.8%. The model was used to investigate the protection afforded by an air-fed, half-face mask. By iteratively changing the dermal exposure concentration, it was possible to predict the ambient concentration that was required to deliver the observed urinary excretion of methylhippuric acid, during and following inhalation exposure to 50 p.p.m. m-xylene vapor. This latter extrapolation demonstrates how physiologically-based pharmacokinetic modeling can be applied in a practical and occupationally relevant way, and permitted a further step not possible with biological monitoring alone. The ability of the model to extrapolate an ambient exposure concentration was dependent upon human metabolism data, thereby demonstrating the mechanistic toxicological basis of model output. The methyl hydroxylation of m-xylene is catalyzed by the hepatic mixed function oxidase enzyme, cytochrome P450 2E1 and is active in the occupationally relevant, ( B100 p.p.m.) exposure range of m-xylene. The use of a scaled-up in vitro maximum rate of metabolism (V(maxc)) in the model also demonstrates the increasingly valuable potential utility of biokinetic data determined using alternative, non-animal methods in human chemical-risk assessment. Panemangalore, M., Dowla, H.A., Byers, M.E., 1999. Occupational exposure to agricultural chemicals: effect on the activities of some enzymes in the blood of farm workers. Int. Arch. Occup. Environ. Health 72 (2), 84–88. Abstract Objective: To determine the effect of different durations of exposure to agricultural chemicals on the activities of the blood enzymes delta-aminolevulinic acid dehydratase (ALAD), superoxide dismutase (SOD), and cholinesterase (ChE) in tobacco field workers. Methods: For this preliminary investigation, eight volunteers (all smoked tobacco) who were working on a small tobacco farm were monitored over a period of 2 years along with a comparable urban unexposed group (n= 4). During the growing season between 1994 and 1996, dermal and respiratory exposure were determined and blood samples were drawn after the following durations of field work: (1) preexposure (0 day); (2) after 1 day of field work (1 day)- workers reentered fields at 24 h after spraying of acephate and maleic hydrazide; (3) after 30 days of field work (postspraying; 30 days); and (4) Postexposure-no tobacco production. Stan-

dard analytical methods were used. Results: Activity of ALAD was depressed by 30% after 1 day and there was no further decrease in ALAD activity after 30 days of field work. SOD activity, in contrast, declined by 29% and 50% after 1 day and 30 days, respectively, as compared with 0-day activity and that of the urban control, which was similar to 0-day activity (P o´ 0.05). Plasma ChE activity declined by 19% after both 1 and 30 days of exposure/field work. The activities of all three enzymes were restored to urban control or preexposure levels during postexposure. Plasma Cd levels were high in the samples taken after 30 days as compared with the preexposure levels. Respiratory nicotine exposure was highest after 30 days of field work. Conclusion: This preliminary study suggests that erythrocyte SOD is a sensitive indicator of exposure to agricultural chemicals in tobacco field workers. Rice, P., Brown, R.F.R., 1999. The development of lewisite vapour induced lesions in the domestic, white pig. Int. J. Exp. Pathol. 80 (1), 59 – 67. Abstract Studies performed in the past in our laboratory have detailed the development of sulphur mustard lesions in the domestic, white pig using small glass chambers to achieve saturated vapour exposure under occluded conditions. We have now used this experimental model to produce cutaneous lesions for detailed histopathological studies following challenge with lewisite. Histological examination of resulting lesions have revealed that although the overall pattern of lesion development is similar to that seen following mustard challenge, the time-course of cellular events is very much compressed. The epidermis showed focal basal-cell vacuolation with associated acute inflammation as early as 1 h postexposure. Coagulative necrosis of the epidermis and papillary dermis was complete by 24 h and followed the appearance of multiple coalescent blisters between 6 and 12 h postexposure. At 48 h, the lesions were full thickness burns with necrosis extending into the deep subcutaneous connective and adipose tissues. The study of lesions beyond 24 h revealed early epithelial regeneration at the wound edge. The overall spontaneous healing rate of these biologically severe lesions was significantly faster than comparable sulphur mustard injuries and probably reflected a lack of alkylation of DNA and RNA. Sawyer, T.W., 1999. Synergistic protective effects of selected arginine analogues against sulphur mustard toxicity in neuron culture. Toxicol. Appl. Pharmacol. 68 (155/2), 169 – 176. Abstract Previous studies in this laboratory have shown that the arginine analogues L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME) have potent protective activity against sulphur mustard (HD) toxicity that was not related to their nitric oxide synthase inhibiting activities. Furthermore, their characteristics of action suggested that they act at different sites to exert their protection. L-TC acted rapidly (minutes of preincubation) and was equipotent in protecting either immature or mature cul-

P.V. Lakshmana Rao et al. / Toxicology 139 (1999) 39–51 tures of chick embryo neurons against the toxicity of HD while L-NAME was only effective in mature cultures. Maximal protection occurred at mM drug concentrations and increased the LC50 of HD by similar 200% (LNAME) to similar 800% (L-TC). L-NAME did not alter the efficacy of L-TC in immature cultures but increased the LC50 up to 1500% in mature cultures. Removal of LNAME eliminated this synergism, leaving only the persistent protection of L-TC. L-nitroarginine and D-NAME also increased the protective efficacy of L-TC in a concentration-related manner in mature cultures. The timing of drug administration before or after HD culture exposure was critical. Drug coadministration resulted in synergistic protection only when L-TC was added to the cultures prior to HD treatment. Thus, synergistic protective effects were also achieved when L-NAME was added up to 8 h after HD exposure, if they were pretreated with L-TC. Based on these findings, it is proposed that HD initiates its toxicity extremely rapidly through a cell surface-mediated event that can be blocked by L-TC. A signal is transduced into the cell that results in an additional event or lesion that manifests itself several hours downstream. This event/lesion progresses to cell death unless blocked reversibly by L-NAME.

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Sawyer, T.W., Risk, D., 1999. Effect of lowered temperature on the toxicity of sulphur mustard in vitro and in vivo. Toxicology 134 (1), 27 – 37. Abstract Primary cultures of chick embryo neurons were exposed to sulphur mustard (HD) and L-nitroarginine methyl ester (L-NAME) and then incubated at either 25 or 37°C. Lowering the temperature of the cultures decreased the 24-h toxicity of HD, but did not increase the efficacy of L-NAME protection. However, the length of time postHD treatment in which L-NAME was maximally effective in protecting against HD toxicity was dramatically enhanced, out to 12 h after HD exposure. In addition, the persistence of L-NAME protection of the cells against HD was significantly lengthened. Tests conducted in human skin keratinocytes also showed that lowering the incubation temperature of actively proliferating, just-confluent or post-confluent cultures significantly and persistently decreased the cytotoxicity of HD. The persistence of LNAME protection was increased in non-proliferating cells. Finally, cooling of HD-vapour exposed sites on hairless guinea pigs for 4.5 h decreased the severity of the resultant lesions out to 72 h post-exposure.