Business Associate Agreements Inventory.

This Business Associate Agreement Inventory is a checklist for use by covered entities who use or disclose individually identifiable health information with or to their Business Associates. The checklist is designed to assist the covered entities to review their contracts with each respective Business Associate and determine whether the contracts contain all of the provisions required by the Health Insurance Portability and Accountability Act regulations. The regulations addressed in the checklist include the Transactions and Code Sets Standards (effective October 16, 2000), the proposed Security Standards, and the final Privacy Regulations (published December 28, 2000).

Rat jejunum controls luminal thiol-disulfide redox.

The control of luminal thiol-disulfide redox state may be important for several intestinal functions, including absorption of iron or selenium and maintenance of mucus fluidity. Disulfides are present in the diet, and although luminal thiols are supplied in bile, little is known about the ability of the small intestine to reduce disulfides to maintain the luminal thiol-disulfide redox state. The objective of the current study was to determine whether the isolated, vascularly perfused jejunum, free from biliary thiols, could reduce intraluminal glutathione disulfide (GSSG) to glutathione (GSH). GSSG was introduced in a deoxygenated solution to inhibit the reoxidation of any GSH formed, and preparations were pretreated with acivicin to inhibit the degradation of GSH by gamma-glutamyltransferase. GSSG (250 micromol/L) was reduced to GSH, with the luminal redox potential (E(h)) for GSSG/2GSH changing from >0 to -111, -132 and -143 mV at 10, 20 and 30 min, respectively. The E(h) for luminal cystine/2cysteine was approximately 20 mV more reducing than that for GSSG/2GSH at each time point, suggesting that cysteine could function in the reduction of GSSG in the lumen. Measurements in specific regions showed that GSSG reduction was more rapid in the duodenum and proximal jejunum than in the distal jejunum. Preparations without acivicin treatment showed that E(h) values were unaffected by inhibition of gamma-glutamyltransferase despite differences in GSH and cysteine pool sizes. Rat intestine has a mechanism to adjust the luminal thiol-disulfide redox. In principle, dysfunction of this mechanism could contribute to malabsorption or other nutritional disorders.

Glutathione S-transferase in mucus of rat small intestine.

Glutathione S-transferases in the small intestine function in detoxification of electrophilic compounds ingested in foods, dietary supplements, and orally administered drug preparations. Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes, the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal concentrations in the rat are about 250 microM. The present study was designed to test the hypothesis that exogenous GSH is used for intestinal conjugation by glutathione S-transferase. The results show that 250 microM of extracellular GSH stimulated conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte preparations. However, an unexpected finding was that most of this stimulated activity did not depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this detoxication enzyme in the extracellular mucus layer provides a novel mechanism for preventing direct contact of potentially toxic dietary electrophiles with the intestinal enterocytes.

Simultaneous measures of contraction and intracellular calcium in single, cultured smooth muscle cells.

Simple methods are presented for quantitating contraction and intracellular calcium simultaneously in single, cultured smooth muscle cells. These methods are the first to demonstrate that reliable velocities of cell shortening can be measured in cultured smooth muscle cells and that cells in vitro exhibit shortening velocities comparable to those measured in the fastest phasic muscles in situ. Temporal relationships between changes in intracellular calcium and shortening within single cells were determined with a resolution of 100 ms and were consistent with measures in more "classical" preparations. Intracellular calcium rose quickly and transiently 10-fold above the basal level of 80-90 nM in response to the muscarinic agonist, carbachol. Shortening of the cells occurred 200 ms after intracellular calcium began to rise. The sensitivity and reliability of these methods allowed the effects of different stimuli to be easily resolved. The present report demonstrates that genuine contractility need not be ignored in cultured smooth muscle cells and that the temporal relations between shortening and intracellular calcium mobilization can be quantitatively assessed in controlled in vitro environments.

Guidelines for the pediatric perioperative anesthesia environment. American Academy of Pediatrics. Section on Anesthesiology.

The American Academy of Pediatrics proposes the following guidelines for the pediatric perioperative anesthesia environment. Essential components are identified that make the perioperative environment satisfactory for the anesthesia care of infants and children. Such an environment promotes the safety and wellbeing of infants and children by reducing the risk for adverse events.

Vitronectin regulates smooth muscle contractility via alphav and beta1 integrin.

Previous work from this laboratory has established a method for maintaining physiological contractility of dissociated avian smooth muscle in a defined medium at low density. The present report emphasizes the dramatic potency of serum to alter smooth muscle phenotype and induce a loss of contractility. Vitronectin, a molecule purified from plasma, mimicked these effects of serum via an integrin that is RGD-sensitive. Studies utilizing blocking antibodies against vitronectin demonstrated that the presence of this specific adhesion molecule was necessary for the serum-induced loss of contractility. Based on the actions of function-blocking antibodies and RGD-containing peptides, the integrin alphavbeta1 appears to be the primary receptor involved in vitronectin's ability to induce phenotypic transformation in amniotic smooth muscle. The influence of vitronectin on smooth muscle contractility is particularly relevant, because this molecule is abundant in whole blood and plasma (approx. 400 microg/ml). The results suggest that smooth muscle needs to be continually protected from normal blood constituents in vivo. The implications of these results for smooth muscle-related diseases like atherosclerosis, restenosis and Kaposi's sarcoma are discussed.

Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Neutrophil- and glutathione-mediated hepatotoxicity of alpha-naphthylisothiocyanate.

In summary, both glutathione and blood neutrophils contribute to ANIT hepatotoxicity. Glutathione contributes by virtue of its ability to form a reversible S-conjugate with ANIT that is critical in shuttling ANIT into bile. Where it is released in large and probably toxic concentrations. The possibility remains that this conjugate may be bioactivated by secondary mechanisms, but no evidence for a toxic glutathionyl conjugate of ANIT currently exists. Neutrophils and platelets both appear to play important roles in ANIT hepatotoxicity. The role of platelets is currently unknown, but studies in vitro raise the possibility that neutrophils may be activated during ANIT exposure to release cytotoxic proteases that cause injury to target cells. Although ANIT activates neutrophils in vitro, the mechanisms by which neutrophils are recruited into the periportal region and activated in vivo remain unknown.

Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.

Leukotrienes and alpha-naphthylisothiocyanate-induced liver injury.

alpha-naphthylisothiocyanate (ANIT) administration to rats results in periportal hepatic inflammation and injury. Glutathione (GSH) appears to be necessary for the liver injury to occur. The leukotrienes (LTs) are metabolites of arachidonic acid and potent mediators of inflammation that have been implicated in certain liver injury models. Inasmuch as GSH is a cofactor for the synthesis of cysteinyl-LTs and since inflammation is a prominent component of ANIT injury, we hypothesized that LTs are involved in producing the hepatic insult that results from ANIT administration. To test this hypothesis, rats were treated with one of several inhibitors of LT biosynthesis, A63162, Zileuton or MK-886. Each of these agents prevented the formation of LTB4 in Ca++ ionophore-stimulated whole blood from rats treated with the inhibitors. A63162 attenuated the hepatic parenchymal injury caused by ANIT and resulted in a modest decrease in ANIT-induced cholestasis. In contrast, neither Zileuton nor MK-886 attenuated liver injury. AT-125 (Acivicin) inhibits gamma-glutamyl transferase (GGT), the enzyme that catalyzes the formation of LTD4 from LTC4. AT-125 pretreatment did not prevent ANIT-induced hepatic parenchymal insult. It did, however, ameliorate the cholestasis caused by ANIT. In conclusion, the partial protection afforded by A63162 and AT-125 likely results from effects unrelated to the formation of LTs, since Zileuton and MK-886 inhibited LT synthesis without affording protection. The lack of protection by Zileuton and MK-886 in the face of LT synthesis inhibition suggests that LTs are not necessary for the expression of injury after ANIT administration.

Clearance of glutathione disulfide from rat mesenteric vasculature.

Organs of the digestive tract, including pancreas, small intestine, and colon, have mechanisms to modulate plasma thiol-disulfide balance. Because plasma glutathione disulfide (GSSG) concentration may be elevated from < 1 microM in control rats to over 25 microM during oxidative stress, we examined whether GSSG was cleared from rat mesenteric vasculature. When 100 microM GSSG was perfused through the gut via the superior mesenteric artery, an average of 45% was lost in a single pass. Results showed that gamma-glutamyltransferase (gamma-GT)-dependent and -independent mechanisms were involved in GSSG loss. Acivicin (AT125) treatment inhibited gamma-GT activity in the mesenteric vasculature by 94% and attenuated the loss of GSSG equivalents by 44%. These results supported a role for gamma-GT in GSSG loss from the mesenteric vasculature but indicated that still other mechanisms were involved in GSSG clearance. Elevations of portal levels of glutathione (GSH) and the mixed disulfide of cysteine and GSH (CySSG) also occurred with vascular GSSG perfusion and could account for about 40% of GSSG equivalents lost. Because portal elevations of GSH and CySSG were not inhibited by AT125, they were formed by a gamma-GT-independent mechanism(s). Given that cysteine was present in the mesenteric vasculature, the most likely mechanism to explain GSH and CySSG formation was via nonenzymatic thiol-disulfide exchange between GSSG and cysteine. Uptake of vascular GSSG by pancreas, small intestine (jejunum and ileum), or colon apparently did not occur as tissue contents of GSSG or GSH were not elevated, except for a small elevation of GSH in pancreas when mesenteric gamma-GT was inhibited with AT125. Additionally, GSSG was not transported from mesenteric vasculature into the small intestinal lumen because luminal levels of GSSG or GSH were not elevated. Further, total cysteine equivalents in lumen were unchanged indicating that GSSG was not transported to lumen and degraded to cystine by gamma-GT and dipeptidases localized to the intestinal brush-border. These results indicate that GSSG present in mesenteric vasculature is metabolized in the vascular compartment by gamma-GT-dependent and -independent reactions; together, these account for over 80% of lost GSSG equivalents. They also suggest that organs of the mesentery may play a quantitatively important role in plasma GSSG clearance and modulation of vascular thiol-disulfide balance.

Secretion of cysteine and glutathione from mucosa to lumen in rat small intestine.

Using a vascularly perfused rat intestinal preparation, we found that large quantities (i.e., 100-200 microM) of acid-soluble thiols accumulated in the jejunal lumen in 10-30 min and that the accumulation was largely unaffected by dietary food restriction for 24 or 48 h. Depending on the length of perfusion, cysteine comprised 20-40% of total luminal thiols, whereas glutathione (GSH) made up only 0-3%. To determine whether luminal cysteine accumulation resulted from mucosal secretion of GSH and subsequent degradation by brush-border gamma-glutamyltransferase (gamma-GT) and dipeptidases, acivicin or serine-borate was used to inhibit gamma-GT. Both agents inhibited gamma-GT activity by > 95%, reduced luminal cysteine by approximately 40-50%, and caused a modest elevation of luminal GSH to approximately 10-13 microM, indicating that GSH secretion does occur but cannot account for all of the luminal cysteine accumulation. Luminal thiol trapping experiments with Ellman's reagent supported this conclusion. Given that cysteine made up 15-20% of the mucosal thiol pool in jejunum, secretion of cysteine from mucosa to lumen likely accounted for the majority of luminal cysteine. Given the mucolytic nature of thiols and the role of cysteine in iron absorption, intestinal thiol secretion may be important in intestinal function.

Maintenance of contractility in dissociated smooth muscle: low-density cultures in a defined medium.

The loss of contractility in long-term cultures of dissociated smooth muscle is such an established observation that the lack of contractility of cultured smooth muscle cells is often not even noted. This report describes methods of dissociating and culturing smooth muscle cells from the avian amnion that maintain contractility for > 1 mo in a defined medium. Because contractility was assessed by monitoring the contractions of individual cells to neurotransmitter-related substances, it is clear that these cells maintained both contractility and pharmacological responsiveness. However, when amniotic smooth muscle cells were dissociated with enzymes containing impurities or cultured in the presence of serum, they flattened and lost contractility, as reported for many other types of smooth muscle.

Extracellular matrix regulates smooth muscle responses to substance P.

Little is known about the extracellular factors that determine a cell's responsiveness to neurotransmitters. This is a particularly important issue for pharmacologically diverse cell types such as neurons and smooth muscle. This report demonstrates that the contractile responses of amniotic smooth muscle to a specific neuropeptide, substance P, is controlled by a molecule(s) intimately associated with the extracellular basement membrane. This molecule(s) normally represses the expression of substance P responsiveness in this tissue. When the amniotic smooth muscle is separated from the basement membrane by dissociation, normally unresponsive cells exhibit a progressive increase in responsiveness to substance P, beginning within the first 24 hr in culture. The induction of substance P responses was completely inhibited when the cells were plated onto isolated amniotic basement membrane rather than onto polyornithine or collagen I. Similar changes in the responsiveness to another agonist, histamine, did not occur. The data demonstrate that extracellular matrix exerts a major instructive influence in determining the responsiveness of avian amniotic smooth muscle to specific ligands. We suggest that similar regulatory mechanisms may operate in other tissues.

Activated neutrophils injure the isolated, perfused rat liver by an oxygen radical-dependent mechanism.

Under certain circumstances, segmented neutrophils (PMNs) injure extrahepatic tissue by releasing toxic oxygen species and degradative enzymes. The authors used an isolated, perfused rat liver preparation to determine whether PMNs might injure the liver. Livers from fasted rats were perfused with Krebs-Ringer bicarbonate buffer (pH 7.4) containing 3% bovine serum albumin (BSA) in a recirculating system. Rat peritoneal PMNs (4 x 10(8] or vehicle (Hank's balanced salt solution [HBSS], pH 7.35) were added, and liver injury was assessed 90 minutes later by release of alanine aminotransferase (ALT) into the perfusion medium and histopathologic analysis of liver sections. Perfusion of livers receiving only HBSS for 90 minutes resulted in a small increase in ALT activity in the perfusion medium but did not significantly alter histologic features of liver sections. Addition of unstimulated PMNs did not increase further the ALT activity and, with the exception of vascular neutrophilia, did not significantly change the histomorphology compared with controls. When PMNs activated with a combination of phorbol myristate acetate (PMA, 31 ng/ml) and lithocholate (100 mumol/l [micromolar]) were added to the perfusion system, however, livers released greater amounts of ALT than those perfused with PMA, lithocholate, and HBSS. Activated PMNs caused a transient reduction in flow of perfusion medium that lasted approximately 5 to 15 minutes. Liver sections had multifocal to coalescing foci of moderate to severe, acute hepatocellular necrosis associated with the areas of intense sinusoidal neutrophilia. In addition a second type of lesion was observed and was characterized by triangular foci of necrosis located adjacent to periportal regions of sinusoids or portal veins containing neutrophilic thrombi. These lesions were void of PMNs and were consistent with infarcts. A combination of superoxide dismutase and catalase added to the perfusion medium (500 U/ml each) prevented the elevation in ALT activity but not the transient reduction in flow. These results indicate that activated PMNs may cause liver injury by an oxygen radical-dependent mechanism. It is unclear whether PMN-derived oxygen radicals, hepatocellular-derived oxygen species resulting from reduced tissue perfusion and reperfusion, or both are involved in the pathogenesis.

Protection against alpha-naphthylisothiocyanate-induced liver injury by decreased hepatic non-protein sulfhydryl content.

alpha-Naphthylisothiocyanate (ANIT) injures bile duct epithelium and hepatic parenchymal cells in rats. It is commonly believed that ANIT must undergo bioactivation by hepatic, cytochrome P450-dependent mixed-function oxidases (MFO), since agents which are inducers or inhibitors of hepatic MFO activity enhance or attenuate, respectively, the liver injury associated with ANIT. Several of these agents also affect hepatic glutathione (GSH) content and/or GSH S-transferase activity in a manner to suggest a causal role for GSH in ANIT-induced hepatotoxicity. To determine whether GSH might be involved in the mechanism of injury, buthionine sulfoximine (BSO), diethyl maleate (DEM), or phorone was used to reduce hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content. Twenty-four hours after ANIT treatment, rats exhibited cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate aminotransferase (AST) activity, and gamma-glutamyltransferase activity. Cotreatment of rats with BSO decreased NPSH content by 70% at 24 hr and prevented the cholestasis and elevations in serum markers of liver injury caused by ANIT. Likewise, cotreatment of rats with DEM afforded protection against markers of liver injury. Phorone treatment attenuated ANIT-induced elevations in serum total bilirubin concentration and AST activity. Although BSO treatment afforded protection against ANIT-induced liver injury at 24 hr, the injury was evident at 48 hr, and it appeared to coincide with a return of hepatic NPSH content. These results suggest that GSH plays a causal or permissive role in the liver injury caused by ANIT.

Relationship between alpha-naphthylisothiocyanate-induced liver injury and elevations in hepatic non-protein sulfhydryl content.

Acute administration of alpha-naphthylisothiocyanate (ANIT) to rats has been used as a model of intrahepatic cholestasis. The mechanism of toxicity of ANIT is unknown, although recent evidence suggests a causal or permissive role for glutathione (GSH) (Dahm LJ and Roth RA, Biochem Pharmacol 42: 1181-1188, 1991). In these studies, ANIT treatment elevated hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content, when liver injury was evident. The purpose of the present study was to characterize the effects of ANIT on hepatic NPSH content and to relate these changes to the development of liver injury. In rats fasted for 24 hr, administration of ANIT (100 mg/kg, per os [p.o.]) did not change hepatic NPSH content, bile flow, or serum measurements of total bilirubin concentration, alanine aminotransferase (ALT) activity, or gamma-glutamyltransferase (GGT) activity by 12 hr post-treatment relative to corn oil vehicle controls. However, by 24 hr after ANIT treatment, rats exhibited cholestasis and elevations in serum markers of liver injury. These markers were associated temporally with an increase in hepatic NPSH content, which consisted entirely of GSH. To determine whether the cholestasis caused by ANIT treatment might have caused the elevation of hepatic NPSH content, an extrahepatic cholestasis in rats was produced by ligation of the common bile duct. Bile duct ligation elevated hepatic NPSH content between 6 and 12 hr after ligation. Administration to rats of a non-hepatotoxic analog of ANIT, beta-naphthylisothiocyanate, also elevated hepatic NPSH content 24 hr after treatment. Taken together, these results indicate that the elevation in hepatic NPSH content after ANIT treatment is associated temporally with the onset of liver injury, but this elevation does not appear to participate causally in the mechanism of injury.

The regulation of synaptogenesis during normal development and following activity blockade.

The mature neuromuscular junction is characterized by the tight spatial colocalization of synaptic vesicles and acetylcholine receptor (AChR) clusters. Although a large body of work exists on the interactions between motoneurons and myotubes leading to synaptogenesis in tissue culture, how the neuromuscular junction acquires its highly specialized structure in vivo is not well understood, particularly during the earliest period of synaptogenesis. In this study, the development of the neuromuscular synapse in chick hindlimb muscles was examined and quantified by simultaneously labeling the pre- and postsynaptic elements from the time the main nerve trunks leave the lumbosacral plexus region to enter the developing limb (St 24) through the end of the motoneuron cell death period (St 36). Based on these results, synaptogenesis can be divided into several distinct stages that are intimately connected to the innervation sequence described in a previous paper (Dahm and Landmesser, 1988). Briefly, as large nerve trunks approach the developing muscles and the first AChR clusters are induced to form on nearby myotubes, none of these initial receptor clusters are in direct contact with a nerve profile. The first appearance of nerve-contacted clusters (synapses) is coincident with the growth of large, unbranched nerve trunks into the muscles. The next step is initiated by the formation of small nerve side branches that grow out from the larger intramuscular nerve trunks to bring most axons and myotubes into contact for the first time. As side branches form, synapses appear around them, and non-nerve-contacted receptor clusters disappear from around the main intramuscular nerve trunks. The next step in synaptogenesis is the restriction of synaptic vesicle antigen to sites of synaptic contact. These early stages of synaptogenesis are also characterized by the growth of the presynaptic terminal to match the length of the postsynaptic receptor cluster. This study showed that AChR cluster formation during early in vivo neuromuscular development does not require close anatomical nerve contact, but that the presence of the nerves is necessary for AChR clusters to form. This suggests that the nerves normally induce AChR clustering via the release of a diffusible substance, a suggestion substantiated by the observation that AChR clusters do not form on aneural myotubes in vivo. In order to assess the role of synapse formation in the regulation of motoneuron number, synaptogenesis was quantitatively examined after chronic neuromuscular blockade, which prevents motoneuron cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

An antibody to neutrophils attenuates alpha-naphthylisothiocyanate-induced liver injury.

alpha-Naphthylisothiocyanate (ANIT) causes cholestasis and injury to bile duct epithelium and hepatic parenchymal cells in rats. The mechanism of toxicity is unknown. Neutrophils (PMNs) infiltrate periportal regions of the liver after ANIT intoxication. Because PMNs play a causal role in other extrahepatic models of tissue injury, we determined whether PMNs might be involved in ANIT-induced liver injury in rats by reducing circulating PMN numbers with a polyclonal antibody (antineutrophil serum). ANIT treatment caused cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate amino-transferase activity, gamma-glutamyltransferase activity and histologic lesions consistent with acute, neutrophilic cholangiohepatitis. Cotreatment of rats with antineutrophil serum reduced circulating PMN numbers, prevented ANIT-induced cholestasis and attenuated other markers of liver injury elevated by ANIT. In addition, antineutrophil serum treatment attenuated the severity of histologic lesions within the liver and reduced the number of PMNs in periportal regions. Numbers of PMNs in liver sections correlated positively with markers of liver injury, histologic evidence of cholangiohepatitis and numbers of circulating PMNs in peripheral blood. The protection afforded by antineutrophil serum appeared to result from a specific reduction of PMNs and not lymphocytes, because administration of an antilymphocyte serum reduced circulating lymphocyte numbers without offering protection. Inasmuch as ANIT stimulates PMNs in vitro to release O2- and since PMN-derived oxygen species may cause tissue injury, we determined whether administration of agents which degrade oxygen radicals afforded protection against the liver injury caused by ANIT.(ABSTRACT TRUNCATED AT 250 WORDS)