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Ultrastructural and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) studies were conducted to compare mechanisms of 2-chloroethyl ethyl sulfide (CEES) and heat-induced injury to EpiDerm. Twenty-two hours after 2-h exposure to the monofunctional alkylating agent CEES, budding of cytoplasm, clumping of nuclear chromatin, disintegration of nuclear membranes and cytoplasmic structures, and cytoplasmic vacuolization were detected, especially in the basal cells near the pseudobasement membrane. TUNEL techniques revealed DNA fragmentation distinct from that normally associated with terminal keratinocyte differentiation. Similar evaluations 22.5 h after 90 min exposure of EpiDerm to elevated temperature (45 degrees C) produced a different pattern of cell damage. Swelling of intercellular spaces, extensive cytoplasmic vacuolization, disruption of normal nuclear shape, reduced cell membrane integrity, and release of cellular material in the basal region characterized heat injury. Heat did not alter the DNA fragmentation normally associated with keratinocyte maturation. These data suggest that CEES elicited an apoptotic mechanism of cell death with features of terminal differentiation such as nuclear membrane disintegration and loss of cytoplasmic structures. Heat, alternatively, produced changes more typical of oncotic necrosis.

Chemical warfare threats require the development of diverse models for the assessment of countermeasures. Human skin products, Skin2 (differentiating keratinocytes on a fibroblast-collagen matrix) and EpiDerm (differentiating keratinocytes) were exposed (2 h) to the sulfur mustard 2-chloroethyl ethyl sulfide (CEES, 1-2 mg l(-1) min(-1)) in humidified air or to humidified air alone. Tissues were evaluated histologically, ultrastructurally and for viability 22 h later; media and tissues were also analyzed for inflammatory mediators. Histology showed that CEES induced the separation of dermal and epidermal regions in Skin2 with severe damage to basal keratinocytes. Histology and electron microscopy of both products revealed condensation of nuclear chromatin, retraction of spinous processes, collapse of the tonofibrillar network and cytoplasmic vacuolization and blebbing in those cells with loss of pseudobasement membrane integrity. Exposure of Skin2 to CEES increased extracellular interleukin-1alpha (IL-1alpha), prostaglandin-E2 (PGE2) and especially IL-1 receptor antagonist (IL-1Ra) release (56,334 vs 84,614 pg ml(-1)), but decreased interleukin-6 (IL-6, 4,755 vs 351 pg ml(-1)). Exposure of EpiDerm to CEES led to unaffected extracellular and reduced intracelluar IL-1alpha (371 vs 92 pg ml(-1)). Extracellular IL-1Ra greatly increased (2,375 vs 24,875 pg ml(-1)), whereas cellular levels decreased (16,5425 vs 96,625 pg ml(-1)). Extracellular (224 vs 68 pg ml(-1)) and intracellular (485 vs 233 pg ml(-1)) soluble interleukin-1 receptor H (sIL-1RII) decreased. Prostanglandin E2 increased (1,835 vs 2,582 pg ml(-1)), whereas heat shock protein 70A (Hsp70A) remained statistically unchanged (57,000 vs 96,000 pg ml(-1)). Failure to obtain a heat shock response to CEES may contribute to the susceptibility of tissue to the alkylating agent. Consistent and marked responses of cellular and extracellular IL-1Ra to CEES suggest a potential for use as a tissue status marker and primary antiinflammatory regulator in skin.

Sulfur mustard provokes an acute inflammatory response in skin. To determine if keratinocytes regulate this response and whether three potential vesicant antagonists can counteract adverse changes, specimens of EpiDerm (MatTek Corp., Ashland, MA), a human skin model of differentiating keratinocytes, were exposed 2 h to humidified air with or without 2-chloroethyl ethyl sulfide (CEES, 1.72-1.73 mg/L/min) with or without 10 mM niacinamide, a poly (ADP-ribose) polymerase (PARP) inhibitor, 25 microM CGS9343B (calmodulin antagonist), or 8.4 mM leupeptin (cysteine protease inhibitor). After a 22-h incubation, levels of interleukin-1 alpha (IL-1alpha), its receptor antagonist (IL-1Ra), soluble type II receptor (sIL-1RII) and prostaglandin-E(2) (PGE(2)) were determined. Methylthiazole tetrazolium (MTT) viability tests and histological observations were also conducted. PGE(2) levels were abundant but unaffected by CEES regardless of antagonist presence. Total amounts (media plus lysate) of IL-1alpha, IL-1Ra, and sIL-1RII were reduced with CEES irrespective of antagonist. CEES promoted the release of IL-1Ra. Exposure of EpiDerm to CEES in the presence of the vesicant antagonists did not improve viability or counteract histological damage. We conclude CEES depresses total IL-1alpha and related cytokines, does not affect PGE(2) release, and adverse changes associated with CEES-exposed EpiDerm are not ameliorated by these particular antagonists. Dramatically increased (5- to 10-fold) release of IL-1Ra may provide a useful marker for cytotoxicity. The high level of IL-1Ra and increased release with injury suggest a primary function in down-regulating IL-1 inflammatory responses in skin.

Living skin equivalent (LSE) was used to identify nonvascular aspects of freeze-thaw injury to human skin cells. Pairs of transwell cell culture inserts, containing LSE, were washed twice for 30 min in maintenance media at 37 degrees C in a CO2 incubator and placed in two wells of a six-well assay tray (containing assay media). One specimen was maintained at room temperature. The other was cooled at 1 degrees C/min to -15 degrees C and rapidly rewarmed. Both were incubated for 24 h on fresh maintenance media at 37 degrees C in a CO2 incubator. This process was repeated producing 12 per group. Prostaglandin E2 (PGE2), interleukin-1 alpha (IL-1 alpha), and K+ were measured (mean +/- SE) in the assay medium, after the rewarming interval, and in the maintenance media after the 24-h incubation. Specimens were then processed for electron microscopy. After the rewarming interval, PGE2 (164.0 +/- 23.2 pg/0.1 ml), IL-1 alpha (24.3 +/- 2.2 pg/0.1 ml), and K+ (6.47 +/- 0.41 mM) released from frozen LSE were significantly increased compared to controls (PGE2 = 22.2 +/- 4.6 pg/0.1 ml, IL-1 alpha = 5.7 +/- 1.0 pg/0.1 ml, K+ = 4.32 +/- 0.02 mM). After the 24-h incubation, PGE2 and IL-1 alpha released by frozen LSE (PGE2 = 1126 +/- 208 pg/0.1 ml; IL-1 alpha = 80.6 +/- 6.8 pg/0.1 ml) remained significantly higher than controls (PGE2 = 229.0 +/- 45 pg/0.1 ml; IL-1 alpha = 4.9 +/- 0.7 pg/0.1 ml). At this time, K+ leakage from frozen LSE (4.39 +/- 0.03 mM) had returned to a normal range (control = 4.65 +/- 0.02 mM). Keratinocytes and, to a lesser extent, fibroblasts showed ultrastructural freeze-thaw damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Several laboratories have demonstrated the value of the isolated perfused rat liver as a suitable model for heat-induced hepatic injury in vivo. Membrane changes caused by perfusion of rat livers at 42 degrees C for 90 min were similar to those induced by toxic chemicals or hypoxia. In an evaluation of several categories of drugs reported to reduce cell injury, calcium antagonists (nifedipine, dantrolene, and verapamil), were evaluated for their therapeutic potential for heat injury. Isolated rat livers were perfused at 42 degrees C for 90 min with and without calcium antagonists. Livers were also perfused at 37 degrees C. Potassium and transaminase leakage, bile production and ultrastructure were used to evaluate their responses. Neither of the three calcium antagonists significantly improved any of the functional parameters measured. However, dantrolene produced dilated or vesicular rough endoplasmic reticulum in the heated livers. These changes suggest selective intracellular action on endoplasmic reticulum of heated livers. Ring-shaped mitochondria and vesicular endoplasmic reticulum were observed in the heated, verapamil-treated livers, but these could not be quantitatively distinguished from controls. Nifedipine did not appear to alter intracellular membranes, but did increase bile production.

Isolated rat livers were perfused at 37 degrees, 41 degrees, 42 degrees, and 43 degrees C with and without insulin and cortisol. Two additional groups were perfused at 42 degrees C with either hormone alone. The perfusate contained red blood cells, amino acids, and albumin in Krebs-Ringer bicarbonate. Bile flow was significantly increased by hormones at 37 degrees C. Bile flow was also increased by hormones at all other temperatures. At 41 degrees C, K+ leakage was the only parameter that indicated injury. Insulin and cortisol significantly reduced K+ leakage at this temperature compared to those without hormones. At 42 degrees C, insulin and cortisol reduced K+ leakage, increased bile flow, reduced transaminase release, and improved ultrastructural integrity. The enhanced bile flow was due primarily to insulin. A reduction in K+ leakage required both insulin and cortisol. Transaminase leakage responded to either hormone alone or in combination; however, only the cortisol-treated group showed a statistically significant reduction in transaminase leakage. At 43 degrees C, indications of irreversible injury were evident and hormones had no beneficial effects. Loss of membrane homeostasis appeared to be the initial event.

Isolated rat livers were perfused for 90 minutes at temperatures from 37 degrees to 43 degrees C. to evaluate the effects of heat alone on bile production, alanine aminotransferase, and asparate aminotransferase release, and light and electron microscopic structure. Bile production reached a plateau after 45 minutes at 43 degrees C. and after 60 minutes at 42 degrees C. At temperatures between 39 degrees and 41 degrees C., bile production was not significantly different from that produced at 37 degrees C. The timing and levels of alanine aminotransferase and aspartate aminotransferase released into the perfusates were similar, with increases after 45 minutes at 43 degrees C., after 60 minutes at 41 degrees and 42 degrees C. and after 75 minutes at 39 degrees and 40 degrees C. At the end of the 90-minute perfusion, light microscopy indicated vacuolization and severe dissociation of hepatocytes at 42 degrees and 43 degrees C., and pronounced centrilobular vacuolization at 41 degrees C. Electron microscopy demonstrated that hepatocytes had sustained extensive damage at 41 degrees to 43 degrees C. Mild focal and probably reversible damage occurred at 39 degrees and 40 degrees C. Since pH and O2 levels were regulated in a nonrecirculating system and perfusion rates were constant, neither acidosis, hypoxia, nor circulatory inadequacy were responsible for the alterations. Therefore, changes were attributed to the direct effects of heat, reflected a continuum from no detectable damage at 37 degrees C. to occasional necrosis of individual cells at 39 degrees to 40 degrees C. and culminated in widespread necrosis at 41 degrees to 43 degrees C. with a 90-minute exposure. These results reflect a time/temperature relationship over a range of temperatures. A hypothesis for the sequence of events in the pathogenesis of heat-induced hepatic injury is described.