Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure.

Bacterial endotoxin [lipopolysaccharide (LPS)] causes liver injury in vivo that is dependent on platelets, neutrophils [polymorphonuclear leukocytes (PMNs)], and several inflammatory mediators, including thrombin. We tested the hypothesis that thrombin contributes to LPS-induced hepatocellular injury through direct interactions with platelets and/or PMNs in vitro. Perfusion of isolated livers from LPS-treated rats with buffer containing thrombin resulted in a significant increase in alanine aminotransferase (ALT) activity in the perfusion medium, indicating hepatocellular damage. This effect was completely abolished by prior depletion of PMNs from the LPS-treated donor rats but not by depletion of platelets, suggesting interaction between thrombin and PMNs in the pathogenesis. Thrombin did not, however, enhance degranulation of rat PMNs in vitro, and it was not directly toxic to isolated rat hepatocytes in the presence of PMNs even after LPS exposure, suggesting that hepatocellular killing by the PMN-thrombin combination requires the intervention of an additional factor(s) within the liver. In livers from naive donors perfused with buffer containing PMNs and LPS, no injury occurred in the absence of thrombin. Addition of thrombin (10 nM) to the medium caused pronounced ALT release. These results indicate that thrombin and PMNs are sufficient extrahepatic requirements for LPS-induced hepatocellular damage in intact liver.

Concurrent inflammation as a determinant of susceptibility to toxicity from xenobiotic agents.

Sensitivity to the toxic effects of xenobiotic agents is influenced by a number of factors. Recent evidence derived from studies using experimental animals suggests that inflammation is one of these factors. For example, induction of inflammation by coexposure to bacterial endotoxin, vitamin A or Corynebacterium parvum increases injury in response to a number of xenobiotic agents that target liver. These agents are diverse in chemical nature and in mechanism of hepatotoxic action. Factors critical to the augmentation of liver injury by inflammation include Kupffer cells, neutrophils, cytokines such as tumor necrosis factor-alpha (TNF-alpha) and lipid mediators such as prostaglandins, but these may vary depending on the xenobiotic agent and the mechanisms by which it alters hepatocellular homeostasis. In addition, the timing of inflammagen exposure can qualitatively alter the toxic response to chemicals. Inflammation-induced increases in susceptibility to toxicity are not limited to liver. Concurrent inflammation also sensitizes animals to the toxic effects of agents that damage the respiratory tract, kidney and lymphoid tissue. It is concluded that inflammation should be considered as a determinant of susceptibility to intoxication by xenobiotic exposure.

Participation of Ca2+/calmodulin during activation of rat neutrophils by polychlorinated biphenyls.

The effects of Ca2+ and Ca2+/calmodulin on the polychlorinated biphenyl (PCB)-induced activation of phospholipase A2 (PLA2) in rat neutrophils were examined. The commercial PCB mixture Aroclor 1242 induced activation of PLA2 and promoted an increase in the intracellular free calcium concentration ([Ca2+]i). Bromoenol lactone (BEL), an inhibitor of the Ca2+-independent PLA2 isoform (iPLA2) activated by PCBs, did not abrogate the increase in [Ca2+]i, suggesting that this change in Ca2+ concentration is not downstream from the activation of iPLA2. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate], a blocker of the release of intracellular Ca2+, decreased Aroclor 1242-induced stimulation of PLA2 with a maximal inhibition of 17% at 50 microM. These two results suggest little direct dependence between the PCB-induced activation of iPLA2 and increase in [Ca2+]i. Calmidazolium and W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], two chemically distinct calmodulin inhibitors, inhibited Aroclor 1242-induced PLA2 activity, whereas trifluoperazine (TFP), another inhibitor of calmodulin, had no effect at noncytotoxic concentrations. Thus, activation of PLA2 is dependent, in part, on calmodulin. Furthermore, both TFP and Aroclor 1242 inhibited neutrophil degranulation stimulated by the bacterial peptide formyl-methionyl-leucyl-phenylalanine. These results raise the possibility that some of the effects of PCBs on neutrophil function can be explained by effects on Ca2+/calmodulin-dependent processes.

Bacterial lipopolysaccharide enhances aflatoxin B1 hepatotoxicity in rats by a mechanism that depends on tumor necrosis factor alpha.

Exposure to a nontoxic dose of bacterial endotoxin (lipopolysaccharide [LPS]) potentiates the hepatotoxicity of aflatoxin B(1) (AFB(1)). Because some of the pathophysiologic effects associated with LPS are mediated through tumor necrosis factor alpha (TNF-alpha), this study was conducted to explore the role of TNF-alpha in the AFB(1)/LPS model. Male Sprague-Dawley rats (250-300 g) were treated with either 1 mg AFB(1)/kg, intraperitoneally, or its vehicle (0.5% dimethyl sulfoxide [DMSO]/water), and 4 hours later with either Escherichia coli lipopolysaccharide (7.4 x 10(6)EU/kg, intravenously) or its saline vehicle. LPS administration resulted in a marked rise in TNF-alpha levels at 6 hours, which preceded the onset of liver injury. TNF-alpha messenger RNA (mRNA) in liver was increased by LPS treatment. The mRNA of receptors (R1 and R2) for TNF-alpha was also examined. R1 mRNA levels were not altered; however, R2 mRNA levels were increased by either AFB(1) or LPS administration. To determine if TNF-alpha plays a causal role in the development of liver injury, the increase in TNF-alpha was attenuated by administration of either pentoxifylline or anti-TNF-alpha serum, and liver injury was assessed. Administration of either of these agents resulted in protection. LPS treatment resulted in the upregulation of gene transcription for cyclooxygenase-2 (COX-2). However, administration of the selective COX-2 inhibitor NS-398 did not decrease injury. TNF-alpha and COX-2 inhibitors did not affect hepatic sequestration of neutrophils. Furthermore, it did not appear that TNF-alpha contributed to injury through inhibition of tissue repair. These data support the hypothesis that LPS-induced expression of TNF-alpha underlies the potentiation of AFB(1)-induced hepatotoxicity.

Lipopolysaccharide augments aflatoxin B(1)-induced liver injury through neutrophil-dependent and -independent mechanisms.

Exposure to small, noninjurious doses of the inflammagen, bacterial endotoxin (lipopolysaccharide, LPS) augments the toxicity of certain hepatotoxicants including aflatoxin B(1) (AFB(1)). Mediators of inflammation, in particular neutrophils (PMNs), are responsible for tissue injury in a variety of animal models. This study was conducted to examine the role of PMNs in the pathogenesis of hepatic injury after AFB(1)/LPS cotreatment. Male, Sprague-Dawley rats (250-350 g) were treated with either 1 mg AFB(1)/kg, ip or its vehicle (0.5% DMSO/saline), and 4 h later with either E. coli LPS (7. 4 x 10(6) EU/kg, iv) or its saline vehicle. Over a course of 6 to 96 h after AFB(1) administration, rats were killed and livers were stained immunohistochemically for PMNs. LPS resulted in an increase in PMN accumulation in the liver that preceded the onset of liver injury. To assess if PMNs contributed to the pathogenesis, an anti-PMN antibody was administered to reduce PMN numbers in blood and liver, and injury was evaluated. Hepatic parenchymal cell injury was evaluated as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum and from histologic examination of liver sections. Biliary tract alterations were evaluated as increased concentration of serum bile acids and activities of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), and 5'-nucleotidase (5'-ND) in serum. Neutrophil depletion protected against hepatic parenchymal cell injury caused by AFB(1)/LPS cotreatment but not against markers of biliary tract injury. This suggests that LPS augments AFB(1) hepatotoxicity through two mechanisms: one of which is PMN-dependent, and another that is not.

Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline.

Individuals are commonly exposed to bacterial endotoxin (lipopolysaccharide [LPS]) through gram-negative bacterial infection and from its translocation from the gastrointestinal lumen into the circulation. Inasmuch as noninjurious doses of LPS augment the hepatotoxicity of certain xenobiotic agents, exposure to small amounts of LPS may be an important determinant of susceptibility to chemical intoxication. Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that at large doses produces centrilobular liver lesions in rats. In the present study, MCT was coadministered with LPS to determine whether LPS would enhance its hepatotoxicity. Doses of MCT (100 mg/kg, ip) and LPS (7.4 x 10(6) EU/kg, iv), which were nonhepatotoxic when administered separately, produced significant liver injury in male, Sprague-Dawley rats when given in combination. Within 18 h after MCT administration, this cotreatment resulted in enhanced plasma alanine aminotransferase and aspartate aminotransferase activities, two markers of liver injury. Histologically, overt hemorrhage and necrosis appeared between 12 and 18 h. The lesions were centrilobular and midzonal and exhibited characteristics similar to lesions associated with larger doses of MCT and LPS, respectively. In the presence of LPS, the threshold for MCT toxicity was reduced to 13-33% of the dose required for toxicity with MCT alone. A study in isolated, hepatic parenchymal cells revealed no interaction between MCT and LPS in producing cytotoxicity. In summary, coexposure of rats to noninjurious doses of MCT and LPS resulted in pronounced liver injury. Results in vitro suggest that the enhanced toxicity does not result from a direct interaction of MCT and LPS with hepatic parenchymal cells. These results provide additional evidence that exposure to small amounts of LPS may be a determinant of susceptibility to food-borne hepatotoxins.

Bacterial lipopolysaccharide exposure augments aflatoxin B(1)-induced liver injury.

Bacterial endotoxin (lipopolysaccharide; LPS) given to animals in large doses results in pronounced, midzonal liver injury. Exposure to smaller, non-injurious doses of LPS augments the toxicity of certain hepatotoxicants. This study was conducted to delineate the development of injury in a rat model of augmentation of aflatoxin B(1) (AFB(1)) hepatotoxicity by LPS. At large doses (i.e., > 1 mg/kg, ip), AFB(1) administration resulted in pronounced injury to the periportal regions of the liver. Male, Sprague-Dawley rats (250-350 g) were treated with 1 mg AFB(1)/kg, ip or its vehicle (0.5% DMSO/saline) and 4 h later with either E. coli LPS (7.4 x 106 EU/kg, iv) or its saline vehicle. Liver injury was assessed 6, 12, 24, 48, 72, or 96 h after AFB(1) administration. Hepatic parenchymal cell injury was evaluated as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum and from histologic examination of liver sections. Biliary tract alterations were evaluated as increased concentration of serum bile acids and activities of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), and 5'-nucleotidase (5'-ND) in serum. At all times and for all markers, injury in rats treated with either AFB(1) or LPS alone was absent or modest. In the AFB(1)/LPS cotreated group, hepatic parenchymal cell injury was pronounced by 24 h and had returned to control values by 72 h. The injury began in the periportal region and spread midzonally with time. Furthermore, changes in serum markers indicative of biliary tract alterations were evident by 12 h and had returned to control values by 72 h. Thus, the nature of the hepatic lesions suggested that LPS potentiated the effects of AFB(1) on both parenchymal and bile duct epithelial cells.

Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity.

Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip). Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS. Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM). These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from naïve rats. These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects.

Activation of neutrophil calcium-dependent and -independent phospholipases A2 by organochlorine compounds.

The production of reactive oxygen species by organochlorine pesticides has been implicated in the toxicity and carcinogenicity of these compounds; however, the mechanism by which these agents stimulate the production of oxygen radicals is unknown. Phospholipase A2 (PLA2)-mediated release of arachidonic acid has been shown to play an essential role in superoxide anion (O2-) production in neutrophils exposed to various physiologic and pharmacologic agents. Therefore, studies were performed to determine if the organochlorine pesticides, lindane and dieldrin, activate neutrophils to produce O2- by a mechanism that requires PLA2. Production of O2- and 3H-AA release increased with similar kinetics and concentration-response relations in neutrophils activated with either dieldrin or lindane. Significant release of 3H-AA was seen in neutrophils stimulated with dieldrin or lindane in calcium-free medium and in the presence of the intracellular calcium chelator BAPTA-AM, suggesting that these agents stimulate a PLA2 that does not require calcium for activation. In addition, both O2- production and 3H-AA release were inhibited in a concentration-dependent manner by BEL, a mechanism-based inhibitor of calcium-independent PLA2. These data suggest that dieldrin and lindane stimulate O2- production by a mechanism that involves PLA2. However, release of 3H-AA was not abrogated completely by BEL nor, in the case of dieldrin, preserved entirely in the absence of calcium. This suggests that more than one isoform of PLA2 is activated by dieldrin and by lindane, and that one isoform is calcium-dependent.

Role of protein phosphorylation in activation of phospholipase A2 by the polychlorinated biphenyl mixture Aroclor 1242.

Polychlorinated biphenyls (PCBs) activate neutrophils to induce degranulation and undergo superoxide production through a mechanism that involves stimulation of phospholipase A(2) (PLA(2)). Since the biochemical processes leading to the PCB-induced activation of this enzyme are unknown, the objective of this study was to determine whether protein phosphorylation has a role in this mechanism. Isolated rat neutrophils were labeled with [(3)H]-arachidonic acid ([(3)H]-AA), and activation of PLA(2) was determined from release of radioactivity into the medium. Exposure to the PCB mixture Aroclor 1242 induced release of [(3)H]-AA, and pretreatment with bromoenol lactone (BEL), an inhibitor of calcium-independent PLA(2), diminished release by 80%. Genistein, an inhibitor of tyrosine kinases, caused a small but significant decrease in Aroclor 1242-stimulated release of [(3)H]-AA. Daidzein, a genistein analog with no activity to inhibit tyrosine kinases, had no effect on [(3)H]-AA release. An inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect Aroclor 1242-induced PLA(2) activity at concentrations selective for p38 MAPK; however, PD 98059, which inhibits MAPK kinase (MEK), decreased [(3)H]-AA release to about the same extent as genistein. Treatment of neutrophils with Aroclor 1242 induced phosphorylation of p44 MAPK, and this phosphorylation was unaffected by BEL but was inhibited by PD 98059. Staurosporine, a nonselective inhibitor of protein kinase C (PKC), inhibited PCB-induced release of [(3)H]-AA. Ro 32-0432, a selective inhibitor of PKC(alpha) and PKC(beta1), produced the greatest degree of inhibition (40%) among the tested protein kinase inhibitors. These results suggest that tyrosine kinases, PKC, and the MEK/MAPK pathway are involved in a fraction of Aroclor 1242-induced activation of PLA(2).

Altered biologic activities of commercial polychlorinated biphenyl mixtures after microbial reductive dechlorination.

The reductive dechlorination of polychlorinated biphenyls (PCBs) by anaerobic bacteria has recently been established as an important environmental fate of these compounds. This process removes chlorines directly from the biphenyl ring with replacement by hydrogen, resulting in a product mixture in which the average number of chlorines per biphenyl is reduced. In this study, dechlorination of commercial PCB mixtures (Aroclors 1242 and 1254) by microorganisms eluted from PCB-contaminated sediments of the River Raisin (Michigan) and Silver Lake (Massachusetts) caused a depletion in the proportion of highly chlorinated PCB congeners and an accumulation of lesser-chlorinated congeners. Dechlorination occurred primarily at the meta and, to a much lesser extent, para positions of biphenyl. The concentrations of the coplanar congeners including 3,3',4,4',5-pentachlorobiphenyl, the most potent dioxinlike congener, were significantly lowered by reductive dechlorination. Microbial reductive dechlorination of commercial PCB mixtures caused a substantial reduction in biologic activities in several instances. It significantly lowered or eliminated the inhibitory effects of Aroclors on fertilization of mouse gametes in vitro. Similarly, the dechlorinated product mixtures had substantially lower ethoxyresorufin-O-deethylase induction potencies and showed less ability to induce activating protein 1 transcription factor activity as compared to the unaltered Aroclors. In other assays the same dechlorinated product mixtures demonstrated biologic activities similar to the nondechlorinated Aroclors, including the ability of PCB mixtures to stimulate insulin secretion and cause neutrophil activation. The data presented here establish that the biologic activities of commercial PCB mixtures are altered by microbial reductive dechlorination and that an assessment of their toxic potential requires an array of tests that include the different mechanisms associated with PCBs.

Effect of three polychlorinated biphenyls on f-met-leu-phe-induced degranulation in rat neutrophils.

It has been known that polychlorinated biphenyl (PCB) mixtures and individual congeners produce degranulation of rat neutrophils. Structure-activity relationships for congeners PCB 8 (2,4'-dichlorobiphenyl), PCB 126 (3,3',4,4',5-pentachlorobiphenyl) and PCB 128 (2,2',3,3',4,4'-hexachlorobiphenyl) were examined by correlating the extent of degranulation and cytotoxicity with molecular and physico-chemical parameters. Neutrophils were exposed to PCB congeners and then to the neutrophil activator f-met-leu-phe (fmlp). Degranulation and cytotoxicity were quantified by measuring released myeloperoxidase and lactate dehydrogenase activities, respectively. Degranulation in the absence of fmlp, that is in quiescent neutrophils, was detected only for PCB 8. Inhibition of fmlp-induced degranulation was observed for both PCB 8 (50 microM) and PCB 128 (10 and 50 microM). PCB 126 did not affect degranulation of quiescent or fmlp-stimulated neutrophils. Thus, effects on degranulation were observed only for ortho-substituted congeners. Cytotoxicity was observed under all conditions with PCB 8, in quiescent neutrophils with PCB 128, and in activated neutrophils with PCB 126. Structure-activity relationships revealed that effects of PCBs on neutrophil degranulation correlate with the energy of the lowest unoccupied molecular orbital but not with torsional angle 2,1,1',2'. This study demonstrates the importance of molecular, electronic parameters in PCB-induced effects on neutrophil degranulation.

Distinct phospholipases A2 regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils.

Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 (PLA2) is important as a substrate for eicosanoid formation and as a second messenger for superoxide anion (O2-) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. Both Aroclor 1242 and A23187 caused release of [3H]AA; however, O2- production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2- production in response to Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Furthermore, calcium-independent, but not calcium-dependent, PLA2 activity was significantly enhanced by Aroclor 1242. These data suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 activates a calcium-independent PLA2 that releases AA for the generation of O2-, and A23187 activates a calcium-dependent PLA2 that mobilizes AA for eicosanoid production.

Symposium overview: toxicity of non-coplanar PCBs.

Research into the mechanism of toxicity of PCBs has focused on the Ah receptor. However, it is becoming increasingly clear that certain ortho-chlorine-substituted, non-coplanar PCB congeners having low affinity for the Ah receptor exhibit important biological activities. Actions of non-coplanar PCB congeners in a variety of biological systems have been discovered and the mechanisms for these effects are being elucidated. The objectives of this symposium are to examine the state of knowledge concerning the mechanisms of toxic action of non-coplanar PCBs and to identify similarities and differences using a variety of biological systems. Effects to be considered will include: neurotoxicity, estrogenicity, insulin release, neutrophil function, calcium regulation, and relevant signal transduction systems. Finally, the symposium addresses the need to consider non-coplanar congeners within the context of risk assessment. The use of Ah-receptor binding and its associated biological effects to assess the total toxicity of PCBs may no longer be defensible because of the actions produced by non-coplanar congeners. This symposium provides documentation for that conclusion and focuses attention on emerging mechanisms of PCB action that have received relatively little attention to date. The topics presented should be of interest to toxicologists interested in mechanisms of action, in PCB risk assessment, and in regulatory toxicology.

Impairment of human neutrophil oxidative burst by polychlorinated biphenyls: inhibition of superoxide dismutase activity.

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O2- to H2O2. Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular O2- production along with a significant decrease in H2O2 production compared with unexposed agonist-stimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L-methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-alpha. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzyme-mediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis.

Neutrophil activation by polychlorinated biphenyls: structure-activity relationship.

Polychlorinated biphenyls (PCBs) rapidly stimulate polymorphonuclear leukocytes (neutrophils) in vitro to produce superoxide anion (O2-). This response results from activation of various intracellular signal transduction pathways and appears to occur in a structure-specific fashion. Individual PCB congeners, varying in pattern and extent of chlorination, were tested for their ability to stimulate production of O2- and/or to enhance the response to protein kinase C activation by phorbol myristate acetate (PMA). Neutrophils were isolated from retired breeder, male, Sprague-Dawley rats and exposed to either vehicle, 10 or 50 microM PCB for 30 min at 37 degrees C. PMA (0 or 20 ng/ml) was added for an additional 10 min, and O2- generated during the incubation period was measured. 2,2'-Dichlorobiphenyl (2,2'-DCB), 2,4'-DCB, or 3,3'-DCB (50 microM) stimulated neutrophils to produce O2-. Incubation of neutrophils with 4,4'-DCB, 3,3',4,4',5-pentachlorobiphenyl (3,3',4,4',5-PeCB), 3,3',4,5,5'-PeCB, or 2,2',3,3',4,4'-hexachlorobiphenyl (2,2',3,3',4,4'-HCB) did not result in generation of O2-. Of the various congeners, 2,4'-DCB elicited the greatest production of O2-. Exposure to 10 microM 2,2'-DCB, 2,4'-DCB, 3,3'-DCB, or 2,2',3,3',4,4'-HCB prior to addition of PMA caused a significant increase in the amount of O2- produced, greater than that seen with either compound alone. PMA-stimulated O2- production was unaffected by prior exposure to 4,4'-DCB, 3,3',4,4',5-PeCB, or 3,3',4,5,5'-PeCB. In separate experiments, 3,3',4,4',5-PeCB inhibited the amount of O2- produced in response to activation with either 3,3'-DCB or 2,4'-DCB. Thus, it appears that congeners which are noncoplanar are capable of stimulating neutrophil O2- production. Coplanar congeners with high affinity for the Ah receptor do not activate neutrophils to produce O2- and may inhibit this response. These results are consistent with the hypothesis that PCBs stimulate neutrophil O2- production by a mechanism that is structure-specific and dependent on the chlorine substitution pattern of the biphenyl rings. Molecular modeling suggested that the sum of atomic charges on chlorine atoms is the most important descriptor for congeners which stimulate O2- production. The angle of rotation and the difference in energy between the highest occupied molecular orbital and the lowest unoccupied molecular orbital are integrative descriptors which, along with the sum of chlorine atomic charges, are associated with this biological activity.

Protein tyrosine kinase involvement in the production of superoxide anion by neutrophils exposed to Aroclor 1242, a mixture of polychlorinated biphenyls.

Neutrophils produce superoxide anion (O2-) when exposed in vitro to Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs). The mechanism for this effect shares some similarities with the mechanism by which the physiologic agonist f-Met-Leu-Phe (fMLP) activates neutrophils. Since production of O2- in response to fMLP involves GTP-binding proteins and protein tyrosine kinases (PTKs), the current study was undertaken to determine whether these signalling pathways are involved in PCB-induced neutrophil activation. Neutrophils exposed to Aroclor 1242 or fMLP produced significant O2-. Pretreatment of intact neutrophils with pertussis toxin or cholera toxin or exposure of permeabilized cells to GDPbetaS significantly inhibited O2- production in fMLP-treated neutrophils but did not alter the response to Aroclor 1242. Pretreatment with genistein, an inhibitor of PTKs, significantly inhibited O2- production in both Aroclor 1242- and fMLP-treated neutrophils; however, daidzein, a structural analogue of genistein which lacks activity against PTKs, was without effect. Exposure of neutrophils to Aroclor 1242 resulted in an increase within 1 min in tyrosine phosphorylation of proteins in the 40 and 60 kDa molecular mass ranges which persisted for up to 10 min. Similar results were obtained with 2,2',4,4'-tetrachlorobiphenyl (2,2',4,4'-TCB), a PCB congener that stimulates O2- production. In contrast, 3,3',4,4',5-pentachlorobiphenyl (3,3',4,4',5-PeCB), a congener that does not generate O2-, caused only a transient increase in tyrosine phosphorylation of proteins in the 40 kDa range with no effect on 60 kDa proteins. These data suggest that Aroclor 1242 activates neutrophils to produce O2- by a mechanism that requires tyrosine kinase activity; however, heterotrimeric G-proteins are not likely to be involved.

Bacterial endotoxin enhances the hepatotoxicity of allyl alcohol.

Lipopolysaccharide (LPS), or bacterial endotoxin, causes liver damage at relatively large doses in rats. Smaller doses, however, may influence the response to other hepatotoxicants. The purpose these studies was to examine the effect of exposure to relatively all doses of LPS on the hepatotoxic response to allyl alcohol, which causes periportal necrosis in laboratory rodents through an known mechanism. Rats were pretreated with LPS (100 micrograms/kg) 2 hr before treatment with a minimally toxic dose of allyl alcohol mg/kg), and liver toxicity was assessed 18 hr later from activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma and from histologic changes in liver sections. Plasma ALT and AST activities were not elevated significantly in rats treated with vehicle, LPS, or allyl alcohol alone, but pronounced increases were observed in rats treated with LPS and allyl alcohol. Significant liver injury occurred as early as 2 hr after allyl alcohol treatment in LPS-pretreated rats and peaked at 6 hr. LPS treatment did not affect the activity of alcohol dehydrogenase and did not affect the rate of production of NADH in isolated livers perfused with allyl alcohol; thus, LPS does not appear to increase the metabolic bioactivation of allyl alcohol into acrolein. On the other hand, pretreatment with 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, abolished the hepatotoxicity of allyl alcohol in LPS-treated rats, indicating that production of acrolein was needed for LPS enhancement of the toxicity of allyl alcohol. Pretreatment of rats with gadolinium chloride (10 mg/kg), a known inactivator of Kupffer cell phagocytic function, decreased LPS augmentation of the response to allyl alcohol. These data indicate that LPS markedly enhances the hepatotoxic response to allyl alcohol. Furthermore, the results suggest that the LPS-induced enhancement of allyl alcohol hepatotoxicity occurs through a Kupffer cell-dependent mechanism.

Alteration by flutamide of neutrophil response to stimulation. Implications for tissue injury.

When activated, inflammatory cells such as polymorphonuclear leukocytes (PMNs) can damage isolated hepatocytes in vitro. These studies were performed to determine if flutamide activates PMNs. Flutamide (Eulexin) is an orally active, nonsteroidal antiandrogen that can cause liver injury associated with inflammation. Activation of PMNs was assessed from the production of superoxide anion and the release of myeloperoxidase in the presence or absence of flutamide and phorbol myristate acetate (PMA) or f-methionyl-leucyl-phenylalanine (fmlp). In addition, hepatocytes were cocultured with PMNs stimulated with PMA or fmlp in the presence or absence of flutamide, and cytotoxicity to hepatocytes was evaluated from the release of alanine aminotransferase into the medium. Flutamide alone did not stimulate the generation of superoxide anion by PMNs but potentiated its production in response to PMA. At lower concentrations of flutamide (i.e. 25 microM), there was a tendency toward increased release of myeloperoxidase, whereas at higher concentrations (i.e. 75-100 microM) flutamide inhibited degranulation in response to fmlp. In coculture with hepatocytes, PMNs exposed to either flutamide, fmlp, or PMA alone caused a significant increase in release of alanine aminotransferase. Hepatocellular toxicity caused by PMNs incubated with flutamide and PMA was additive and was not affected by the addition of superoxide dismutase and catalase. Flutamide had no significant effect on fmlp-induced injury in cocultures. These data indicate that flutamide alters the activation of PMNs by subsequent stimuli in vitro. In addition, in the presence of flutamide, minor PMN-mediated injury to isolated hepatocytes was observed.

Gadolinium chloride pretreatment protects against hepatic injury but predisposes the lungs to alveolitis after lipopolysaccharide administration.

Exposure to lipopolysaccharide (LPS) can result in multi-organ failure and death. After an intravenous injection of LPS into rats, neutrophils (PMN) rapidly accumulate in the liver sinusoids and pulmonary vasculature, and PMN play a critical role in producing both hepatic and pulmonary injury. Kupffer cells (KC), the resident macrophages of the liver, phagocytose LPS and produce inflammatory mediators which may be chemotactic and stimulatory for PMN. The purpose of this study was to determine whether inhibition of KC function affects PMN accumulation and the development of parenchymal injury in the liver and lungs after systemic administration of LPS. Female, Sprague-Dawley rats (180-230 g) were pretreated with either gadolinium chloride-6H2O (GdCl3; 10 mg/kg, intravenously), to inactivate KC, or saline vehicle 24 h before receiving either LPS (4 mg/kg, intravenously) or saline vehicle. Rats were killed 1.5, 6, and 24 h after LPS administration. In a preliminary study, exposure to GdCl3 decreased uptake of carbon in the liver, indicating inhibition of phagocytosis by KC. Ninety minutes after administration of LPS, PMN accumulated in the livers of LPS-treated rats, and this effect was not altered by pretreatment with GdCl3. Similarly, exposure to LPS resulted in PMN accumulation in the pulmonary tissue, which was unaffected by GdCl3 pretreatment. Exposure to GdCl3 before LPS administration resulted in a significant increase in the number of PMN recovered by bronchoalveolar lavage at 24 h, indicating diffuse acute alveolitis. LPS-induced hepatic injury was prevented by pretreatment with GdCl3; however, the increased wet lung/body weight ratio observed after LPS administration was unaffected by GdCl3. These results confirm that inactivation of KC protects against hepatic injury and extend this finding by ruling out inhibition of hepatic PMN accumulation as a mechanism for this effect. The data also suggest that treatment with GdCl3 predisposes the lungs to alveolitis during systemic exposure to LPS.