90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Characterization and pathogenesis of aerosolized eastern equine encephalitis in the common marmoset (Callithrix jacchus).

BACKGROUND: Licensed antiviral therapeutics and vaccines to protect against eastern equine encephalitis virus (EEEV) in humans currently do not exist. Animal models that faithfully recapitulate the clinical characteristics of human EEEV encephalitic disease, including fever, drowsiness, anorexia, and neurological signs such as seizures, are needed to satisfy requirements of the Food and Drug Administration (FDA) for clinical product licensing under the Animal Rule. METHODS: In an effort to meet this requirement, we estimated the median lethal dose and described the pathogenesis of aerosolized EEEV in the common marmoset (Callithrix jacchus). Five marmosets were exposed to aerosolized EEEV FL93-939 in doses ranging from 2.4 × 101 PFU to 7.95 × 105 PFU. RESULTS: The median lethal dose was estimated to be 2.05 × 102 PFU. Lethality was observed as early as day 4 post-exposure in the highest-dosed marmoset but animals at lower inhaled doses had a protracted disease course where humane study endpoint was not met until as late as day 19 post-exposure. Clinical signs were observed as early as 3 to 4 days post-exposure, including fever, ruffled fur, decreased grooming, and leukocytosis. Clinical signs increased in severity as disease progressed to include decreased body weight, subdued behavior, tremors, and lack of balance. Fever was observed as early as day 2-3 post-exposure in the highest dose groups and hypothermia was observed in several cases as animals became moribund. Infectious virus was found in several key tissues, including brain, liver, kidney, and several lymph nodes. Clinical hematology results included early neutrophilia, lymphopenia, and thrombocytopenia. Key pathological changes included meningoencephalitis and retinitis. Immunohistochemical staining for viral antigen was positive in the brain, retina, and lymph nodes. More intense and widespread IHC labeling occurred with increased aerosol dose. CONCLUSION: We have estimated the medial lethal dose of aerosolized EEEV and described the pathology of clinical disease in the marmoset model. The results demonstrate that the marmoset is an animal model suitable for emulation of human EEEV disease in the development of medical countermeasures.

Transient lipopolysaccharide-induced resistance to aerosolized Bacillus anthracis in New Zealand white rabbits.

Previous studies have demonstrated that prior infection by various bacterial pathogens induces nonspecific resistance to subsequent infection by other gram-negative and gram-positive bacterial pathogens. In the present study, we evaluated whether underlying inflammation enhanced host resistance to inhalational Bacillus anthracis infection in New Zealand White rabbits (SPF; Bordetella- and Pasteurella-free). Accordingly, rabbits were pretreated with either the inflammagen bacterial LPS (60,000 EU/kg), a component of the outer membrane of gram-negative bacteria, or saline (vehicle). Administration of LPS resulted in brief pyrexia and a significant increase in the proinflammatory cytokine TNFα, thus confirming LPS-induced inflammation. At 24 h after LPS treatment, rabbits were exposed to aerosolized B. anthracis spores (Ames strain; approximately 300 LD50). Blood samples collected at various times after challenge were cultured. Compared with their saline-pretreated counterparts, LPS-pretreated, B. anthracis challenged rabbits exhibited delays in 2 biomarkers of B. anthracis infection-anthrax-induced pyrexia (25 h versus 66 h after challenge, respectively) and bacteremia (26 h versus 63 h, respectively)-and survived longer (41 h versus 90 h, respectively). Similar to control animals, all LPS-pretreated, B. anthracis-challenged rabbits exhibited pathology consistent with inhalational anthrax. Taken together, these results suggest that prior or underlying stimulation of the innate immune system induces transient host resistance to subsequent B. anthracis infection in SPF New Zealand white rabbits. In particular, our results emphasize the importance of using animals that are free of underlying infections to prevent confounding data in studies for inhalational anthrax characterization and medical countermeasure evaluation.

Endogenous interleukin-4 regulates glutathione synthesis following acetaminophen-induced liver injury in mice.

In a recent study, we reported that interleukin (IL)-4 had a protective role against acetaminophen (APAP)-induced liver injury (AILI), although the mechanism of protection was unclear. Here, we carried out more detailed investigations and have shown that one way IL-4 may control the severity of AILI is by regulating glutathione (GSH) synthesis. In the present studies, the protective role of IL-4 in AILI was established definitively by showing that C57BL/6J mice made deficient in IL-4 genetically (IL-4(-/-)) or by depletion with an antibody, were more susceptible to AILI than mice not depleted of IL-4. The increased susceptibility of IL-4(-/-) mice was not due to elevated levels of hepatic APAP-protein adducts but was associated with a prolonged reduction in hepatic GSH that was attributed to decreased gene expression of γ-glutamylcysteine ligase (γ-GCL). Moreover, administration of recombinant IL-4 to IL-4(-/-) mice postacetaminophen treatment diminished the severity of liver injury and increased γ-GCL and GSH levels. We also report that the prolonged reduction of GSH in APAP-treated IL-4(-/-) mice appeared to contribute toward increased liver injury by causing a sustained activation of c-Jun-N-terminal kinase (JNK) since levels of phosphorylated JNK remained significantly higher in the IL-4(-/-) mice up to 24 h after APAP treatment. Overall, these results show for the first time that IL-4 has a role in regulating the synthesis of GSH in the liver under conditions of cellular stress. This mechanism appears to be responsible at least in part for the protective role of IL-4 against AILI in mice and may have a similar role not only in AILI in humans but also in pathologies of the liver caused by other drugs and etiologies.

Aerosolized Bacillus anthracis infection in New Zealand white rabbits: natural history and intravenous levofloxacin treatment.

The natural history for inhalational Bacillus anthracis (Ames strain) exposure in New Zealand white rabbits was investigated to better identify potential, early biomarkers of anthrax. Twelve SPF Bordetella-free rabbits were exposed to 150 LD(50) aerosolized B. anthracis spores, and clinical signs, body temperature, complete blood count, bacteremia, and presence of protective antigen in the blood (that is, antigenemia) were examined. The development of antigenemia and bacteremia coincided and preceded both pyrexia and inversion of the heterophil:lymphocyte ratio, an indicator of infection. Antigenemia was determined within 1 h by electrochemiluminescence immunoassay, compared with the 24-h traditional culture needed for bacteremia determination. Rabbits appeared clinically normal until shortly before succumbing to anthrax approximately 47 h after challenge or approximately 22 h after antigenemia, which suggests a relatively narrow therapeutic window of opportunity. To evaluate the therapeutic rabbit model, B. anthracis-exposed rabbits were treated (after determination of antigenemia and later confirmed to be bacteremic) intravenously with the fluoroquinolone antibiotic levofloxacin for 5 d at a total daily dose of 25 or 12.5 mg/kg, resulting in nearly 90% and 70% survival, respectively, to the study end (28 d after challenge). The peak level for 12.5 mg/kg was equivalent to that observed for a 500-mg daily levofloxacin dose in humans. These results suggest that intravenous levofloxacin is an effective therapeutic against inhalational anthrax. Taken together, our findings indicate that antigenemia is a viable and early biomarker for B. anthracis infection that can be used as a treatment trigger to allow for timely intervention against this highly pathogenic disease.

Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury.

Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2(-/-) mice were treated with 300mg APAP/kg, 90% of JNK2(-/-) mice died of ALF compared to 20% of WT mice within 48h. The high susceptibility of JNK2(-/-) mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair. Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered.

Hepatoprotective role of endogenous interleukin-13 in a murine model of acetaminophen-induced liver disease.

Recent evidence suggests that a deficiency in one or more hepatoprotective regulatory mechanisms may contribute to determining susceptibility in drug-induced liver disease. In the present study, we investigated the role of interleukin (IL)-13 in acetaminophen (APAP)-induced liver disease (AILD). Following APAP (200 mg/kg) administration to male C57BL/6 wild-type (WT) mice, hepatotoxicity developed up to 24 h post-APAP, with a concomitant increase in serum IL-13 concentration. Pretreatment of these mice with an IL-13-neutralizing antibody exacerbated liver injury, as did APAP administration to IL-13 knockout (KO) mice in comparison to WT mice. No difference was observed in either overall APAP-protein adduct formation or liver glutathione levels between KO and WT mice following APAP administration, suggesting that the increased susceptibility of IL-13 KO mice to AILD was not due to enhanced APAP bioactivation but rather injurious downstream events. In this regard, multiplex antibody arrays were used to identify potential IL-13-regulated biomarkers, including various cytokines and chemokines, as well as nitric oxide (NO), associated with AILD that were present at higher concentrations in the sera of APAP-treated IL-13 KO mice than in WT mice. Subsequent inhibition studies determined interferon-gamma, NO, neutrophils, natural killer cells, and natural killer cells with T-cell receptors had pathologic roles in AILD in IL-13 KO mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor modulating the susceptibility to AILD and may provide hepatoprotection, in part, by down-regulating protoxicant factors and cells associated with the innate immune system.

Recurrence of chromosome 10 Thiel-Behnke corneal dystrophy (CDB2) after excimer laser phototherapeutic keratectomy or penetrating keratoplasty.

PURPOSE: To evaluate the recurrence of Thiel-Behnke dystrophy (linked to the 10 q23-q24 locus) after phototherapeutic keratectomy or penetrating keratoplasty. METHODS: This is a retrospective study of 4 patients (8 eyes) who underwent phototherapeutic keratectomy and 1 patient (2 eyes) who underwent penetrating keratoplasty. Best corrected visual acuity was assessed, and biomicroscopic examinations for evidence of recurrent dystrophy were documented and photographed. The location, lesion distribution, and lesion pattern of any recurrence was noted. RESULTS: Follow-up ranged from 8 months to 25 years (mean +/- SD 9.7 +/- 7.97 years). All 10 eyes showed biomicroscopic evidence of central recurrence. Six eyes showed an intermediate zone of honeycomb opacities as well as a peripheral zone of focal and geographic lesions. Despite the high incidence of recurrence, functional central visual acuity was maintained. All eyes maintained functional best corrected visual acuity (ranging from 20/25 to 20/80) despite the postoperative recurrence. CONCLUSION: Recurrence of Thiel-Behnke corneal dystrophy is extremely high after either phototherapeutic keratectomy or penetrating keratoplasty. Despite the high incidence of recurrence, the central cornea is the last to be affected. The peripheral-to-central progression of the lesions points to an epithelial origin for the pathogenesis of the dystrophy. Phototherapeutic keratectomy in the treatment of Thiel-Behnke corneal dystrophy offers a safe and effective treatment modality, providing patients up to 8 years of improved vision ranging from 8 months to 8 years (mean +/- SD 3.7 +/- 2.7 years) and delaying or circumventing the need for more invasive intraocular surgical intervention.

Prediction of corneal haze using an ablation depth/corneal thickness ratio after laser epithelial keratomileusis.

PURPOSE: To investigate the usefulness of ablation depth/corneal thickness (AD/CT) ratio to predict corneal haze after laser epithelial keratomileusis (LASEK) using a retrospective, comparative, interventional case series. METHODS: Fifty patients (90 eyes; mean age 40.9 years) with myopia, hyperopia, and/or astigmatism underwent bilateral or unilateral LASEK for correction of refractive error. After epithelial flaps were created using an 18% alcohol solution, bilateral or unilateral LASEK was performed using the Alcon Autonomous LADARVision 4000 excimer laser. Visual acuity (best spectacle-corrected and uncorrected) and refractive error were measured before and after LASEK. Corneas were assessed by two independent evaluators under a slit-lamp biomicroscope with broad tangential illumination. The relative haze scale was quantitated: 0 (clear), 0.5+ (trace), 1+ (mild), 2+ (moderate), 3+ (marked), and 4+ (severe). RESULTS: Mean preoperative spherical equivalent refraction was -5.46 +/- 3.74 D (range -12.375 to +5.00 D), mean ablation depth was 93.04 +/- 45.03 microm (range 21.2 to 207.2 microm), and mean AD/CT ratio was 0.18 +/- 0.09 (range 0.04 to 0.41). Of 90 eyes, 40 eyes had a higher ablation depth (AD/CT ratio > 0.18) and 50 eyes had a lower ablation depth (AD/CT ratio < 0.18); 92.5% of eyes in the higher ratio group developed clinically significant haze (1+ or greater). In the lower ratio group, 94% of eyes developed no more than 1+ corneal haze, if any. CONCLUSION: The ablation depth/corneal thickness ratio is useful for predicting corneal haze after LASEK. An AD/CT ratio of 0.18 or more suggests that patients have a high risk of developing clinically significant haze (1+ or more) after LASEK.

Update on laser subepithelial keratectomy (LASEK).

PURPOSE OF REVIEW: This study reviews current concepts in laser subepithelial keratectomy (LASEK), variations in LASEK techniques, the role of pharmacology in LASEK, and optimizing outcomes in LASEK. RECENT FINDINGS: Recent studies continue to support the use of LASEK over that of LASIK in the correction of refractive error. In addition, the advent of pharmacological/biologic intervention, improved algorithms, and wavefront technology have expanded the armamentarium available to ophthalmologists in the maximization of LASEK outcomes. SUMMARY: LASEK offers an excellent profile in terms of both final outcome (uncorrected visual acuity) and safety (best corrected visual acuity). Untoward effects of LASEK are readily prevented/treated with a variety of agents. Postoperative pain can be ameliorated using topical and oral analgesia. Infection can be most effectively addressed with the fourth generation of fluoroquinolones. Haze may be treated or prevented using numerous remedies namely autologous serum, steroids, ascorbic acid, mitomycin-c, and NSAIDS. Wavefront combined with LASEK rather than with LASIK may offer the best refractive outcome.

Exclusion of the human collagen type XVII (COL17A1) gene as the cause of Thiel-Behnke corneal dystrophy (CDB2) on chromosome 10q23-q25.

PURPOSE: Determination of the gene causing Thiel-Behnke Corneal Dystrophy (CDB2) would have important clinical implications. Previous studies in our laboratory have suggested that the COL17A1 gene may be the cause of Thiel-Behnke Corneal Dystrophy (CDB2) on Chromosome 10q23-q25. METHODS: We evaluated a five-generation family with CDB2 mapped to chromosome 10. Many of these family members were diagnosed by slit-lamp microscopy. In addition, genomic DNA was isolated and purified from peripheral blood samples. The COL17A1 gene was screened for possible disease causing mutations by PCR and DNA sequencing analysis. RESULTS: No disease-causing mutations were found in any of the 56 exons of the COL17A1 gene or in any of the flanking intron/exon junctions. CONCLUSIONS: Mutations in the coding sequence of the human collagen XVII (COL17A1) gene are not the cause of CDB2.

Cleavage of corneal basement membrane components by ethanol exposure in laser-assisted subepithelial keratectomy.

PURPOSE: To determine the anatomic cleavage plane after exposure to 20% ethanol for approximately 20 to 25 seconds to create an epithelial flap in laser-assisted subepithelial keratectomy (LASEK). SETTING: Ocular Surface Research & Education Foundation, Miami, Florida, and Hermann Eye Center Refractive Surgery Center, Houston, Texas, USA. METHODS: Immunofluorescence staining using monoclonal antibodies against laminin 5, collagen VII, and integrins beta(1) and beta(4) was performed to determine the anatomic location of the cleavage plane in an epithelial flap created by 20-second exposure to 20% ethanol in cadaver eyes and in epithelial flaps obtained from LASEK patients. RESULTS: Immunofluorescence staining to laminin 5 and integrin beta(4) was patchy in the lifted flap and the remaining corneal basement membrane. Immunostaining to collagen VII, the main component of anchoring fibrils, remained exclusively in the corneal bed. Immunostaining to integrin beta(1), present in the pericellular location of all epithelial cell layers, remained exclusively in the epithelial flap. This finding was consistent in cadaver corneas and LASEK epithelial flaps. CONCLUSIONS: The cleavage plane of the ethanol-induced corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane, where integrin beta(4) interacts with laminin 5 to form hemidesmosomes.

Endothelial cell injury and coagulation system activation during synergistic hepatotoxicity from monocrotaline and bacterial lipopolysaccharide coexposure.

A small, noninjurious dose of bacterial lipopolysaccharide (LPS; 7.4 x 106 EU/kg) administered 4 h after a small, nontoxic dose of monocrotaline (MCT; 100 mg/kg) produces synergistic hepatotoxicity in rats within 6 to 12 h after MCT exposure. The resulting centrilobular (CL) and midzonal (MZ) liver lesions are characterized by hepatic parenchymal cell (HPC) necrosis. Pronounced hemorrhage, disruption of sinusoidal architecture, and loss of central vein intima suggest that an additional component to injury may be the liver vasculature. In the present investigation, the hypothesis that sinusoidal endothelial cell (SEC) injury and coagulation system activation occur in this model was tested. Plasma hyaluronic acid (HA) concentration, a biomarker for SEC injury, was significantly increased in cotreated animals before the onset of HPC injury and remained elevated through the time of maximal HPC injury (i.e., 18 h). SEC injury was confirmed by immunohistochemistry and electron microscopy. Pyrrolic metabolites were produced from MCT by SECs in vitro, which suggests that MCT may injure SECs directly through the formation of its toxic metabolite, monocrotaline pyrrole. Inasmuch as SEC activation and injury can promote hemostasis, activation of the coagulation system was evaluated. Coagulation system activation, as marked by a decrease in plasma fibrinogen, occurred before the onset of HPC injury. Furthermore, extensive fibrin deposition was observed immunohistochemically within CL and MZ regions after MCT/LPS cotreatment. Taken together, these results suggest that SEC injury and coagulation system activation are components of the synergistic liver injury resulting from MCT and LPS coexposure.

The coagulation system contributes to synergistic liver injury from exposure to monocrotaline and bacterial lipopolysaccharide.

Coexposure to a noninjurious dose of bacterial lipopolysaccharide (LPS; 7.4 x 106 EU/kg) and a nontoxic dose of the food-borne toxin monocrotaline (MCT; 100 mg/kg) leads to synergistic hepatotoxicity in Sprague-Dawley rats. Inflammatory factors, such as Kupffer cells (KCs), tumor necrosis factor-alpha (TNF)-alpha, and neutrophils (polymorphonuclear leukocytes; PMNs), are critical to the pathogenesis. Inasmuch as activation of the coagulation system and sinusoidal endothelial cell (SEC) injury precede hepatic parenchymal cell (HPC) injury, and since fibrin deposition occurs within liver lesions, the coagulation system might be a critical component of injury. In this study, this hypothesis is tested, and the interdependence of the coagulation system and inflammatory factors is explored. Administration of the anticoagulants heparin or warfarin to MCT/LPS-cotreated animals attenuated HPC and SEC injury. Morphometric analysis revealed that anticoagulant treatment significantly reduced the area of centrilobular and midzonal lesions. Heparin treatment also reduced fibrin deposition in these regions. Furthermore, anticoagulant treatment decreased hepatic PMN accumulation but did not affect plasma TNF-alpha concentration. Neither KC inactivation nor TNF-alpha depletion prevented activation of the coagulation system. PMN depletion, however, prevented coagulation system activation, suggesting that PMNs are needed for this response. These results provide evidence that the coagulation system and its interplay with PMNs are important in the pathogenesis of MCT/LPS-induced liver injury.

Role of neutrophils in the synergistic liver injury from monocrotaline and bacterial lipopolysaccharide exposure.

Synergistic liver injury develops in Sprague-Dawley rats from administration of a small, noninjurious dose (7.4 x 10(6) EU/kg) of bacterial lipopolysaccharide (LPS) given 4 h after a nontoxic dose (100 mg/kg) of the pyrrolizidine alkaloid, monocrotaline (MCT). Previous studies demonstrated that liver injury is mediated through inflammatory factors, such as Kupffer cells and tumor necrosis factor alpha (TNF-alpha), rather than through simple interaction between MCT and LPS. In the present study, the hypothesis that neutrophils (polymorphonuclear leukocytes or PMNs) are causally involved in this injury model is tested, and the interdependence between PMNs and other inflammatory components is explored. Hepatic PMN accumulation and the appearance of cytokine-induced neutrophil chemoattractant-1 in plasma preceded the onset of liver injury, suggesting that PMNs contribute to toxicity. Hepatic PMN accumulation was partially dependent on TNF-alpha. Prior depletion of PMNs in MCT/LPS-cotreated animals resulted in attenuation of both hepatic parenchymal cell (HPC) and sinusoidal endothelial cell (SEC) injury at 18 h. PMN depletion did not, however, protect against early SEC injury that occurred before the onset of HPC injury at 6 h. This observation suggests that SEC injury is not entirely dependent on PMNs in this model. In vitro, MCT caused PMNs to degranulate in a concentration-dependent manner. These results provide evidence that PMNs are critical to the HPC injury caused by MCT/LPS cotreatment and contribute to the progression of SEC injury.

The role of Kupffer cells and TNF-alpha in monocrotaline and bacterial lipopolysaccharide-induced liver injury.

Coexposure to small, noninjurious doses of the pyrrolizidine alkaloid phytotoxin monocrotaline (MCT) and bacterial lipopolysaccharide (LPS) results in synergistic hepatotoxicity. Both centrilobular and midzonal liver lesions occur and are similar to those seen from large, toxic doses of MCT and LPS, respectively. The nature of the lesions in vivo and results from studies in vitro suggest that injury is mediated indirectly rather than from a simple interaction of MCT and LPS with hepatic parenchymal cells. Accordingly, the role of inflammatory factors, such as Kupffer cells and TNF-alpha, in the development of MCT/LPS-induced liver injury was investigated. In Sprague-Dawley rats, MCT (100 mg/kg, i.p.) was administered 4 h before LPS (7.4 x 10(6) EU/kg, i.v.). Pretreatment of these animals with gadolinium chloride, an inhibitor of Kupffer cell function, attenuated liver injury 18 h after MCT administration. An increase in plasma TNF-alpha preceded the onset of hepatic parenchymal cell injury, raising the possibility that this inflammatory cytokine contributes to toxicity. Either pentoxifylline, an inhibitor of cellular TNF-alpha synthesis, or anti-TNF-alpha serum coadministered to MCT/LPS-treated animals significantly attenuated liver injury. These results suggest that Kupffer cells and TNF-alpha are important mediators in the synergistic hepatotoxicity resulting from MCT and LPS coexposure.

The temporal relationship between bacterial lipopolysaccharide and monocrotaline exposures influences toxicity: shift in response from hepatotoxicity to nitric oxide-dependent lethality.

Liver injury from a variety of hepatotoxicants, including the food-borne phytotoxin monocrotaline (MCT), can be augmented by exposure to a noninjurious dose of the inflammagen bacterial lipopolysaccharide (LPS). In a previous study, a nontoxic dose of LPS given 4 h after MCT resulted in synergistic hepatotoxicity within 12-18 h. This study was designed to determine whether temporal differences in MCT and LPS exposure affect toxicity. When LPS (3.4 x 10(6) EU/kg; iv) was given one hour before MCT (100 mg/kg; ip), hepatotoxicity developed between 4 and 8 h after MCT administration, and mortality was much greater than when LPS was administered 4 h after MCT. To explore this difference, the temporal relationship between LPS and MCT exposure (7.4 x 10(6) EU/kg and 100 mg/kg, respectively) was altered. Twenty-four-hour survival was high in animals that received LPS 4 h before (86%) or after (88%) MCT, but it decreased markedly when LPS was administered 1 h before MCT (17%). Using this latter dosing regimen, animals became moribund as early as 4 h after MCT administration. Since liver injury was similar from regimens that differed greatly in mortality, death appeared to result from extrahepatic causes. To explore a role for nitric oxide (NO)-induced shock in this regimen, animals were treated with aminoguanidine (AG), an inhibitor of inducible NO synthase, prior to administration of LPS given an hour before MCT. In the cotreated animals, AG significantly attenuated mortality and decreased plasma nitrate/nitrite concentrations, markers of NO biosynthesis. Hence, the primary target of toxicity from MCT and LPS cotreatment appeared to shift from the liver to an extrahepatic site or sites as exposure to these agents occurred closer together temporally. NO appears to be causally involved in the deaths of animals treated with LPS 1 h before MCT.