A cutaneous full-thickness liquid sulfur mustard burn model in weanling swine: clinical pathology and urinary excretion of thiodiglycol.

Sulfur mustard (bis(2-chloroethyl)sulfide, HD) is a well-known blistering chemical warfare agent. We have developed a cutaneous full-thickness HD burn model in weanling pigs for efficacy testing of candidate treatment regimens. This report addresses clinical pathology findings and the urinary excretion profile of a major HD metabolite (thiodiglycol, TDG) in this model. Six female Yorkshire pigs were exposed to HD liquid on the ventral surface for 2 h, generating six 3-cm diameter full-thickness dermal lesions per pig. Blood samples were collected throughout a 7-day observation period for hematology and serum chemistry examinations. Urine was collected in metabolism cages. Routine urinalysis was performed and the urine analyzed for TDG using gas chromatography/mass spectrometry. Examination of clinical pathology parameters revealed subtle HD-related changes that are suggestive of a mild hemolytic episode. No other signs of clinically significant systemic toxicities were noted, including bone marrow suppression. Thiodiglycol was detected at the earliest time point tested (6-8 h post-exposure) at levels ranging from 0.66 to 4.98 microg ml(-1) with a mean of 2.14 microg ml(-1). Thiodiglycol concentrations were the highest for half of the animals at this earliest time point and at 24-48 h for the others. By the evening of day 3, the mean level had reached 50 ng ml(-1). Mean levels remained 10-40 ng ml(-1) for the remainder of the 7-day observation period, with the highest individual concentration noted during this period of 132 ng ml(-1). Our results are in general agreement with the TDG excretion profiles previously described for rodent models and humans. Urinary excretion of absorbed HD in our weanling pig wound healing model appears to follow the same pattern as is seen in other laboratory animals models. In general, urinary excretion of TDG appears to peak within the first 1-4 days following exposure, with detectable levels after 1 week. Relatively high urinary TDG levels may thus indicate agent exposure within the previous 96 h. Low levels significantly above natural background levels may indicate either exposure to low levels of agent or exposure that occurred more than 4 days prior to collection of the sample.

Posttreatment with eicosatetraynoic acid decreases lung edema in guinea pigs exposed to phosgene: the role of leukotrienes.

Acetylenic acids such as 5,8,11,14-eicosatetraynoic acid (ETYA), have been shown to be effective in preventing pulmonary edema formation (PEF). In phosgene-exposed guinea pigs, we examined the effects of ETYA on PEF, measured as real time lung weight gain (lwg). Pulmonary artery pressure (Ppa), airway pressure (Paw), perfusate leukotrienes (LT) C4/D4/E4/B4, and lung tissue lipid peroxidation (TBARS) were measured using the isolated, buffer-perfused lung model. Guinea pigs were challenged to 175 mg/m3 (44 ppm) phosgene for 10 minutes giving a concentration x time product of 1750 mg.min/m3 (437 ppm.min). Five minutes after removal from the exposure chamber, guinea pigs were treated, i.p., with 200 microL of 100 microM ETYA. 200 microL of 50 microM ETYA was added to the perfusate every 40 minutes, beginning at 60 minutes after start of exposure (t = 0). There were four groups in this study: air-treated, phosgene-exposed, ETYA-posttreated + phosgene, and ETYA-posttreated + air ETYA-posttreated + phosgene guinea pigs had significantly lower Ppa (P = .006), Paw (P = .009), and lwg (P = .016) compared with phosgene-exposed animals. Phosgene exposure reduced LTB4 compared with air-treated controls (P = .09). ETYA-posttreatment + phosgene had significantly increased perfusate LTB4 (P = .0006) compared with phosgene exposure only group. Total perfusate, LTC4 + LTD4 + LTE4, was not different between phosgene-exposed, air-treated or ETYA-posttreatment + phosgene over time. Posttreatment with ETYA significantly lowered TBARS formation, 206 +/- 13 versus 285 +/- 23 nmol/mg protein (P = .016), compared with phosgene-exposed lungs. Paradoxically, ETYA posttreatment decreased PEF and lipid peroxidation, but increased sulfidopeptide LT release from the lung during perfusion. We conclude that LTC4/D4/E4, and B4, may play different roles than previously thought for PEF in the isolated perfused lung model.

Distribution of tritiated dihydromicrocystin in swine.

The distribution of tritiated dihydromicrocystin [3H]2H-MCLR was studied in anesthetized specific-pathogen-free pigs. Two doses were administered i.m. and one dose was given via an isolated ileal loop. At 4 hr after i.v. administration of the toxin at 25 micrograms/kg, 64.6% of the total dose (%TD) was located in the liver, with smaller amounts distributed to the kidneys (1.2% TD), lungs (1.75% TD), heart (0.22% TD), ileum (0.13% TD) and spleen (0.04% TD). A similar distribution was found at 4 hr postdosing in pigs given 75 micrograms/kg, although the liver contained a lower fraction of the total dose, at 46.99% TD, and the kidneys had somewhat more, at 2.19% TD, than the low dose. At the high dose, the fractions of the amount given accounted for by the lungs (0.55% TD), heart (0.23% TD), ileum (0.20% TD) and spleen (0.07% TD) were similar to those at the low dose. The livers of the pigs given 75 micrograms/kg via the ileal loop, at 5 hr postdosing, contained 49.5% TD and the ileum had 33.94% TD. Smaller amounts were distributed to kidneys (1.04% TD), lungs (0.65% TD), heart (0.81% TD) and spleen (0.16% TD). The livers of both groups dosed at 75 micrograms/kg contained higher concentrations of toxin, but lower percentages of the total dose, than the livers of pigs dosed at 25 micrograms/kg. Larger increases in serum arginase in the two 75 micrograms/kg groups were associated with histological evidence of more severe liver damage than at the 25 micrograms/kg dose. Analysis of radiolabeled compounds from hepatic tissue using fast atom bombardment mass spectrometry determined that the primary constituent was [3H]2H-MCLR, but two minor radioactive components were also isolated. These findings indicate that [3H]2H-MCLR is rapidly concentrated in the liver of swine, whether given i.v. or via an isolated ileal loop, that at extremely toxic doses uptake is slowed, and that it is as toxicologically active as the parent compound.

Toxicokinetics of tritiated dihydromicrocystin-LR in swine.

The toxicokinetics of tritiated dihydromicrocystin-LR ([3H]2H-MCLR) were studied in anesthetized, specific-pathogen-free pigs. Pigs were dosed with radiolabeled plus non-labeled 2H-MCLR at 25 or 75 micrograms/kg i.v., or via an isolated ileal loop at 75 micrograms/kg. The i.v. doses were rapidly removed from the blood. At either i.v. dose, more than half the radiolabel from [3H]2H-MCLR present in the blood at 1 min postdosing was cleared by 6 min. The blood clearance at the 75 micrograms/kg dose was slower than at the 25 micrograms/kg dose. Accordingly, at the high dose, the concentrations of the toxin in blood were disproportionately higher from 10 min after dosing until the study ended 4 hr later. The decreased clearance is presumably due to decreased elimination from the blood as a consequence of the hepatic injury that was observed histologically. Following administration of [3H]2H-MCLR at 75 micrograms/kg via the ileum, the maximal toxin concentration in blood was achieved at 90 min after dosing. At that time the [3H]2H-MCLR concentration in portal venous blood was 3.6 times higher than in peripheral venous blood. Although bile production varied, following i.v. dosing radioactivity was detected in bile as early as 12 min postdosing in one animal. This study demonstrated that [3H]2H-MCLR is rapidly removed from the blood of anesthetized swine and that excretion of the radiolabel into bile may begin within 30 min of dosing.

Changes in absorbance at 413 nm in plasma from three rodent species exposed to phosgene.

Mice, rats and guinea pigs were exposed to phosgene (COCl2), a highly irritating and oxidizing gas. Animals were exposed to 87 mg/m3 phosgene for 20 min in a whole-body exposure chamber. Within 55-65 minutes after the start of exposure, plasma was scanned spectrophotometrically from 200-600 nm. A distinct and significant increase in area under the curve in the Soret band region at 413 nm was observed in plasma from phosgene-exposed animals when compared with air-exposed controls in all three species. These peaks were consistent with hemoglobin, an indication that the integrity of the erythrocyte membrane had been compromised by exposure. An erythrocyte osmotic fragility assay on blood from mice exposed to phosgene indicated that 30% less NaCl was needed to cause 50% hemolysis compared to air-exposed mice. These results suggest a new mechanism of phosgene-induced acute lung injury that may be linked, in part, to a direct attack on the erythrocyte membrane.

Efficacy of ibuprofen and pentoxifylline in the treatment of phosgene-induced acute lung injury.

Phosgene, a highly reactive former warfare gas, is a deep lung irritant which produces adult respiratory distress syndrome (ARDS)-like symptoms following inhalation. Death caused by phosgene involves a latent, 6-24-h, fulminating non-cardiogenic pulmonary edema. The following dose-ranging study was designed to determine the efficacy of a non-steroidal anti-inflammatory drug, ibuprofen (IBU), and a methylxanthine, pentoxifylline (PTX). These drugs were tested singly and in combination to treat phosgene-induced acute lung injury in rats. Ibuprofen, in concentrations of 15-300 mg kg-1 (i.p.), was administered to rats 30 min before and 1 h after the start of whole-body exposure to phosgene (80 mg m-3 for 20 min). Pentoxifylline, 10-120 mg kg-1 (i.p.), was first administered 15 min prior to phosgene exposure and twice more at 45 and 105 min after the start of exposure. Five hours after phosgene inhalation, rats were euthanized, the lungs were removed and wet weight values were determined gravimetrically. Ibuprofen administered alone significantly decreased lung wet weight to body weight ratios compared with controls (P < or = 0.01) whereas PTX, at all doses tested alone, did not. In addition, the decrease in lung wet weight to body weight ratio observed with IBU+PTX could be attributed entirely to the dose of IBU employed. This is the first study to show that pre- and post-treatment with IBU can significantly reduce lung edema in rats exposed to phosgene.

Structural modifications imparting reduced toxicity in microcystins from Microcystis spp.

A cyanobacterial (blue-green algal) bloom containing Microcystis aeruginosa (dominant), M. viridis, and M. wesenbergii, was collected from Homer Lake (Illinois, U.S.A.) in the summer of 1988 and microcystins were isolated. One microcystin of substantially reduced toxicity was isolated, together with ten hepatotoxic microcystins. The compound with reduced toxicity was nonlethal at 1 mg/kg (i.p. mouse) and was determined to have a (C3H7O2) mono-ester of the alpha-carboxyl on the Glu unit of microcystin-LR. The other nine microcystins apart from MCLR had approximate LD50S ranging from 97 micrograms/kg to 750 micrograms/kg.

Autoionization reaction of phosgene (OCCl2) studied by electron paramagnetic resonance/spin trapping techniques.

The reaction of phosgene with nitrone spin traps was investigated using electron paramagnetic resonance (EPR)/spin trapping techniques. Evidence for the intermediacy of a carbamoyl monochloride intermediate was obtained. Isotopic substitution of 13C-phosgene was employed to verify the hyperfine coupling constant assignments. The implications of these observations on pulmonary damage caused by inhalation of phosgene are mentioned.