Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens.

Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative cells or spores is advisable.

Fate of Salmonella Senftenberg in broiler chickens evaluated by challenge experiments.

Experimental and epidemiological evidence has indicated the respiratory route to be a potential portal of entry for salmonellas in poultry. The purpose of this study was to evaluate and compare the infectivity of Salmonella enterica serovar Senftenberg following oral gavage, intratracheal or intravenous challenge in chickens. Seven-day-old chicks were challenged with either 10(4) or 10(6) colony-forming units of S. Senftenberg per chick by oral gavage, intratracheal or intravenous challenge, respectively, in two independent trials. Chickens were humanely killed 24 h post challenge and S. Senftenberg was cultured and enumerated from caecal contents, caecal tissue-caecal tonsils and liver and spleen. In both trials, intratracheal delivery of S. Senftenberg was the only route that allowed colonization of the caeca of chickens when compared with oral gavage or intravenous challenge in a dose response fashion (P < 0.05). Liver and spleen samples yielded no S. Seftenberg after the lower dose challenge by the oral or intratracheal route and only low levels following the high-dose administration by these routes, whereas intravenous challenge resulted in recovery of the organisms after both doses. The results of the present study suggest that S. Senftenberg entering the blood is likely to be cleared and will not be able to colonize caeca to the same extent as compared with intratracheal challenge. Clarification of the potential importance of the respiratory tract for transmission of salmonellas under field conditions may be of critical importance to develop intervention strategies to reduce the transmission in poultry.

Evaluation of the respiratory route as a viable portal of entry for Salmonella in poultry via intratracheal challenge of Salmonella Enteritidis and Salmonella Typhimurium.

Experimental and epidemiological evidence suggests that primary infection of Salmonella is by the oral-fecal route for poultry. However, the airborne transmission of Salmonella and similar enteric zoonotic pathogens has been historically neglected. Increasing evidence of Salmonella bioaerosol generation in production facilities and studies suggesting the vulnerabilities of the avian respiratory architecture together have indicated the possibility of the respiratory system being a potential portal of entry for Salmonella in poultry. Presently, we evaluated this hypothesis through intratracheal (IT) administration of Salmonella Enteritidis and Salmonella Typhimurium, as separate challenges, in a total of 4 independent trials, followed by enumeration of cfu recovery in ceca-cecal tonsils and recovery incidence in liver and spleen. In all trials, both Salmonella Enteritidis and Salmonella Typhimurium, challenged IT colonized cecae to a similar or greater extent than oral administration at identical challenge levels. In most trials, chickens cultured for cfu enumeration from IT-challenged chicks at same dose as orally challenged, resulted in an increase of 1.5 log higher Salmonella Enteritidis from ceca-cecal tonsils and a much lower dose IT of Salmonella Enteritidis could colonize ceca to the same extent than a higher oral challenge. This trend of increased cecal colonization due to IT challenge was observed with all trails involving week-old birds (experiment 2 and 3), which are widely considered to be more difficult to infect via the oral route. Liver-spleen incidence data showed 33% of liver and spleen samples to be positive for Salmonella Enteritidis administered IT (10(6) cfu/chick), compared with 0% when administered orally (experiment 2, trial 1). Collectively, these data suggest that the respiratory tract may be a largely overlooked portal of entry for Salmonella infections in chickens.

Effect of different concentrations of acetic, citric, and propionic acid dipping solutions on bacterial contamination of raw chicken skin.

Bacterial contamination of raw, processed poultry may include spoilage bacteria and foodborne pathogens. We evaluated different combinations of organic acid (OA) wash solutions for their ability to reduce bacterial contamination of raw chicken skin and to inhibit growth of spoilage bacteria and pathogens on skin during refrigerated storage. In experiment 1, raw chicken skin samples were dipped into a suspension of either 10(8) cfu/mL of Salmonella Typhimurium, Escherichia coli O157:H7, or Listeria monocytogenes for 30 s and then immersed in PBS or an OA wash solution mixture of 0.8% citric, 0.8% acetic, and 0.8% propionic acid (at equal wt/vol concentrations) for an additional 30 s. In experiment 2, three different concentrations of the OA wash solution (0.2, 0.4, and 0.6% at equal wt/vol concentrations) were tested against chicken skin samples contaminated with Salmonella Typhimurium. Viable pathogenic bacteria on each skin sample were enumerated after 1 and 24 h of storage at 4°C in both experiments. In experiment 3, skin samples were initially treated on d 1 with PBS or 2 concentrations of the OA mixture (0.4 and 0.8%), and total aerobic bacteria were enumerated during a 2-wk storage period. In all experiments, significant (P < 0.05) differences were observed when skin samples were treated with the OA wash solution and no spoilage organisms were recovered at any given time point, whereas increasing log10 numbers of spoilage organisms were recovered over time in PBS-treated skin samples. These results suggest that 0.2 to 0.8% concentrations of an equal-percentage mixture of this OA combination may reduce pathogens and spoilage organisms and improve food safety properties of raw poultry.

Evaluation of selected direct-fed microbial candidates on live performance and Salmonella reduction in commercial turkey brooding houses.

As effective probiotic Bacillus isolates that can increase BW gain (BWG) are identified, they may offer advantages in terms of stability, cost, and feed application over probiotics limited to drinking water application. Additionally, an effective direct-fed microbial (DFM) may offer an effective alternative to antibiotic growth promoters. Previously, 4 Bacillus isolates were identified and evaluated in our laboratory as potential DFM candidates. These isolates were shown to significantly increase BWG as well as reduce recovery of Salmonella after experimental infection. In the first experiment, isolates PHL-MM65 (a Bacillus laterosporus) and PHL-NP122 (a Bacillus subtilis) were evaluated using poults raised under commercial conditions. After 7 d of conventional brooding, poults were tagged, weighed, and placed in 1 of 4 replicate pens for each treatment group [negative control, 0.019% nitarsone, PHL-MM65 (10(6) spores/g of feed), or PHL-NP122 (10(6) spores/g of feed)] within the commercial turkey barn. At 23 d, poults were weighed and BW was calculated. Treatment with PHL-NP122 (853 g) or nitarsone (852 g) increased BW (P ≤ 0.05) compared with control (784 g), whereas treatment with PHL-MM65 (794 g) did not significantly improve BW. Also on d 23 of the trial, ceca were aseptically removed from 10 poults per pen and cultured for recovery of Salmonella. Both Bacillus isolates PHL-NP122 and PHL-MM65 resulted in a significant reduction (P ≤ 0.05) in the frequency of Salmonella by more than 25% compared with the controls. In a second experiment on a different farm, isolates PHL-NP122, PHL-RW33 (a B. subtilis), and PHL-B1 (a Bacillus licheniformis) were evaluated. None of the candidate Bacillus DFM or the group fed nitarsone had significantly different BW or BWG than untreated control. These data suggest that isolate PHL-NP122, when added as a DFM to turkey diets, may increase BW gain as well as nitarsone during the brooding phase of commercial turkey production.

Evaluation of Bacillus species as potential candidates for direct-fed microbials in commercial poultry.

Increasing sociopolitical concerns with antibiotic use have led to investigations of potential alternatives for food safety and growth promotion. Direct-fed microbials (DFM) including spore-based probiotics are amenable to feed inclusion and are extremely stable. We isolated several Bacillus spp. from environmental and poultry sources and tested them for their ability to reduce Salmonella in vitro. In a preliminary in vivo trial, day-of-hatch chicks and poults were randomly assigned to the following treatments (24 birds/treatment): control and one of 8 DFM candidates at 10(6) spores/g of feed. Chicks and poults were tagged, weighed, and orally challenged with Salmonella Typhimurium (ST). Body weight gain and ST recovery were measured 11 d posthatch. Total percentages of ST-positive crop and ceca were significantly lower (P < 0.05) in at least 3 DFM candidates compared with control. Additionally, beneficial effects on BW gain were observed in at least 5 DFM candidates (P < 0.05) compared with control. In a second study, birds treated with NP122 (identified as Bacillus subtilis) had significantly lower (P < 0.05) cecal ST than control and benefitted BW gain irrespective of the presence or absence of a Salmonella challenge. In conclusion, NP122 markedly reduced ST recovery and increased BW gain in both chicks and poults. This provides preliminary evidence that this isolate may have potential use as a DFM in poultry.

Evaluation of Salmonella-vectored Campylobacter peptide epitopes for reduction of Campylobacter jejuni in broiler chickens.

Campylobacter is a leading cause of bacterial gastroenteritis in humans and is often linked to contaminated poultry products. Live Salmonella vectors expressing three linear peptide epitopes from Campylobacter proteins Cj0113 (Omp18/CjaD), Cj0982c (CjaA), and Cj0420 (ACE393) were administered to chicks by oral gavage on the day of hatch, and the chicks were challenged with Campylobacter jejuni on day 21. All three candidate vaccines produced consistent humoral immune responses with high levels of serum IgG and mucosal secretory IgA (sIgA), with the best response from the Cj0113 peptide-expressing vector. Campylobacter challenge following vaccination of three candidate vaccine groups decreased Campylobacter recovery from the ileum compared to that for controls on day 32. The Cj0113 peptide-expressing vector reduced Campylobacter to below detectable levels. The Salmonella-vectored Cj0113 subunit vaccine appears to be an excellent candidate for further evaluation as a tool for the reduction of Campylobacter in poultry for improved food safety.

Development and evaluation of candidate recombinant Salmonella-vectored Salmonella vaccines.

Attenuated Salmonella Enteriditis (ΔSE) recombinant vaccine vectors incorporating a Salmonella flagellar filament protein (fliC) subunit, a putative cell-mediated epitope, for expression of the lamB gene (encoding a maltose outer membrane porin), with or without co-expression of a putative immune-enhancing CD154 oligopeptide, were developed and compared with wild-type Salmonella Enteriditis (experiments 1 and 2) or the attenuated ΔSE empty vector (experiment 3) as initial vaccine candidates against Salmonella infection. A total of 3 experiments were performed to assess the infection and clearance rate of each of these constructs. Each construct or Salmonella Enteriditis was orally administered to broiler chicks at day of hatch by oral gavage (~10(8) cfu/chick). In experiments 1 to 3, liver-spleen and cecal tonsils were removed aseptically for recovery of wild-type Salmonella Enteriditis or ΔSE mutants. These experiments suggested that cell surface expression of fliC alone markedly increased the clearance rate of the vector at or before 21d postvaccination in all 3 experiments. In a fourth experiment, broilers were vaccinated with one of the vaccine constructs or the ΔSE empty vector and then challenged with wild-type Salmonella Typhimurium. At 19 d posthatch, 16 d postinfection, neither candidate protected against challenge significantly better than the ΔSE empty vector, although there was significantly less Salmonella recovered from vaccinated chickens as compared with nonvaccinated controls. These experiments indicate that these experimental vaccines did not protect against heterologous challenge or enhance clearance after Salmonella Typhimurium challenge; as such, their value as vaccines is limited. The increased clearance of the candidate vaccines, particularly the vector expressing fliC alone, may have value in that the fliC epitope may decrease the clearance time of other recombinant vectored Salmonella vaccines.

Investigation of mitochondrial protein expression and oxidation in heart muscle in low and high feed efficient male broilers in a single genetic line.

The objective of this study was to assess the expression of mitochondrial proteins and oxidized proteins in heart muscle homogenate obtained from male broilers exhibiting either high or low feed efficiency (G:F) phenotypes. Tissue homogenate was prepared from ventricular tissue obtained from broilers with high (0.80+/-0.01, n=8) and low (0.62+/-0.02, n=8) FE. The levels of specific immunoreactive proteins and oxidized proteins (carbonyls) were determined using Western blot analysis. The expression of 6 electron transport chain proteins [complex II, 70S subunit (CII 70S); iron-sulfur-containing protein (ISP), cytochrome b (Cyt b), cytochrome (Cyt c1) (of complex III); and cytochrome oxidase subunit II (COX II) (of complex IV)] and adenine nucleotide translocator 1 (ANT1) were higher in low feed efficiency heart mitochondria, but 1 protein [NAD subunit 6c (NAD6c) (complex I)] was higher in high feed efficiency birds. Protein carbonyl levels, indicative of oxidized proteins, were higher in heart tissue of low compared with high feed efficiency broilers.

Differential expression of mitochondrial and extramitochondrial proteins in lymphocytes of male broilers with low and high feed efficiency.

Studies were conducted to investigate relationships between mitochondrial and extramitochondrial protein expression, and protein oxidation in lymphocytes obtained from broilers in which individual feed efficiencies were obtained. Lymphocytes were isolated from male broilers from a single line that were shown to exhibit either low (0.48 +/- 0.02, n = 8) or high (0.68 +/- 0.01, n = 7) feed efficiency (FE). Western blot analysis showed that, compared with lymphocytes from high FE broilers, lymphocytes from low FE broilers exhibited a) higher amounts of oxidized proteins (protein carbonyls), b) lower amounts of 3 mitochondrial proteins [core I, cyt c 1 (complex III), and ATP synthase (complex V)], and c) higher amounts of 2 proteins [30 S (complex II) and COX II (complex IV)]. Two-dimensional gel electrophoresis revealed that the intensities of 25 protein spots from pooled samples of lymphocytes from high and low FE broilers differed by 5-fold or more. Three of these protein spots were picked from the gel and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. One protein spot of ~33 kDa was tentatively identified by MALDI-TOF as a fragment of collapsin-2, a component of semaphorin 3D. The results of this study provide further evidence of increased oxidation associated with low FE and further evidence of differential protein expression associated with the phenotypic expression of feed efficiency.

Feed efficiency and mitochondrial function.

Studies have been conducted in our laboratory to assess differences in mitochondrial function and biochemistry in male broilers with high and low feed efficiency (FE) from the same genetic line and fed the same diet. Mitochondria obtained from broilers with low FE exhibited greater uncoupling of the electron transport chain (ETC) that was apparently due to site-specific defects in electron transport resulting in higher amounts of reactive oxygen species (ROS) compared with high FE mitochondria. Higher amounts of ROS production in Low FE mitochondria were likely responsible for higher protein carbonyl levels, indicative of higher protein oxidation compared with High FE mitochondria and tissue. In turn, higher protein damage in Low FE mitochondria may have contributed to lower activity of electron transport chain complexes relative to values observed in high FE mitochondria. Low FE mitochondria did not exhibit a compromised ability to carryout oxidative phosphorylation, and although there were differences in expression of certain electron transport chain proteins, there was nothing that would indicate that differences in coupling and respiratory chain activity could be due to a general decrease in protein expression between low and high FE mitochondria. The results of these studies provide insight into understanding cellular mechanisms associated with the phenotypic expression of feed efficiency in broilers.

Compromised liver mitochondrial function and complex activity in low feed efficient broilers are associated with higher oxidative stress and differential protein expression.

Variations in broiler growth and efficiency have been explained in part by differences in mitochondrial function and biochemistry in broilers. To further our knowledge in this regard, 2 experiments were carried out to determine the relationships of a) mitochondrial function and activities of various electron transport chain (ETC) complexes; b) production of H2O2, a reactive oxygen species (ROS), and its association with protein oxidation; and c) mitochondrial protein expression in liver of a single line male broilers with low or high feed efficiency (FE, n = 5 to 8 per group). Mitochondrial function and complex activities were measured polarographically and spectrophotometrically, respectively. H2O2 was measured fluorimetrically, whereas oxidized protein (carbonyls) and specific mitochondrial proteins were analyzed using Western blots. Mitochondrial function (ETC coupling) and activities of ETC complexes (I, II, III, and IV) were higher in high FE compared with low FE broilers. H2O2 and protein carbonyls were higher in the livers of low FE broilers than in high FE broilers. Whereas the expression of 4 immunoreactive proteins [NAD3 (complex I), subunit VII (complex III), cytochrome c oxidase subunits (COX) II, and COX IVb (complex IV)] were higher in low FE liver mitochondria and 2 proteins [subunit 70 (complex II) and a-ATP synthase (complex V)] were higher in high FE birds, there were no differences between groups in the expression of 18 other mitochondrial proteins. In conclusion, increases in oxidative stress in low FE broilers were caused by or may contribute to differences in mitochondrial function (ETC coupling and complex activities) or the differential expression of steady-state levels of some mitochondrial proteins in the liver. Understanding the role of oxidative stress in Low FE broilers will provide clues in understanding the cellular basis of feed efficiency.

Biochemical evaluation of mitochondrial respiratory chain in duodenum of low and high feed efficient broilers.

Increased H2O2 production, indicating higher oxidative stress, and lower mitochondrial function was previously observed in duodenal mitochondria isolated from broilers with low feed efficiency (FE, gain:feed). Thus, experiments were conducted to 1) evaluate the activity of the respiratory chain complexes (complexes I to V) and 2) assess protein oxidation and mitochondrial protein expression in broilers with low and high FE. Duodenal mitochondria were isolated from broiler breeders with low (0.52 +/- 0.01) and high (0.68 +/- 0.01) FE (n = 8/group). Respiratory chain complex activities were measured spectrophotometrically, whereas mitochondrial protein expression and protein oxidation (carbonyls) were assessed with Western blots. The activities of all complexes, except complex IV, were lower in the low FE compared with high FE mitochondria, whereas protein carbonyl levels were higher in low FE mitochondria. Steady-state levels of 6 out of 7 nuclear-encoded respiratory chain subunits [70S(FP), core I, core II, cytochrome c (cyt c)1, iron-sulfur protein (ISP), and ATPase-alpha] were higher, whereas 3 out of 6 mitochondrial-encoded subunits (ND4, ND6-C, and COX II) were lower in the low FE group, suggesting that sensitivity of mitochondrial proteins to H2O2 or oxidation varies. The general reduction in complex activity and differential protein expression concomitant with higher oxidized proteins in low FE mitochondria suggest that oxidative stress could be contributing to the lower mitochondrial function observed in low FE duodenal mitochondria.

Low feed efficient broilers within a single genetic line exhibit higher oxidative stress and protein expression in breast muscle with lower mitochondrial complex activity.

The objectives of this study were to determine the effects of low or high feed efficiency (FE) on a) protein oxidation, b) the activities of various respiratory chain complexes, and c) expression of various mitochondrial proteins in male broilers within a single genetic line. Tissue homogenate or mitochondria were isolated from breast muscle of broilers with high (0.80 +/- 0.01) and low FE (0.62 +/- 0.02). The complex activities were measured spectrophotometrically, and the levels of oxidized protein (carbonyl) and immunoreactive mitochondrial proteins were analyzed using Western blots. Protein carbonyl levels were higher in low FE compared with high FE broilers breast muscle, which indicated enhanced protein oxidation in low FE mitochondria. Activities of all respiratory chain complexes (I, II, III, IV) were higher in high FE compared with low FE broilers for breast mitochondria. Whereas the expression of immunoreactive proteins was higher in low FE muscle mitochondria for 5 mitochondrial proteins [core I, cyt c1, cyt b (complex III), COX II (cytochrome c oxidase subunit II, complex IV), and adenine nucleotide translocator (ANT1)], there were no differences between groups in the expression of 9 other respiratory chain protein subunits associated with complexex I, II, III, IV, and V. SDS-PAGE revealed a protein band of 47 kDa that was expressed at a higher level in low FE compared with high FE mitochondria. The differential expression of certain mitochondrial proteins and the 47-kDa band might be a compensatory response either to the lower complex activities or increased protein oxidation observed in low FE birds.

Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity.

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

Measurement of acetaminophen-protein adducts in children and adolescents with acetaminophen overdoses. .

Acetaminophen-protein adducts are biomarkers of acetaminophen toxicity present in the centrilobular region of the liver of laboratory animals following the administration of toxic doses of acetaminophen. These biomarkers are highly specific for acetaminophen-induced hepatic injury and correlate with hepatic transaminase elevation. The objective of this prospective, multicenter study was to evaluate the clinical application of the measurement of acetaminophen-protein adducts in pediatric acetaminophen overdose patients. Serum samples were obtained from 51 children and adolescents with acetaminophen overdose at the time of routine blood sampling for clinical monitoring. Six subjects developed "severe" hepatotoxicity (transaminase elevation > 1,000 IU/L), and 6 subjects had transaminase elevation of 100 to 1,000 IU/L. Acetaminophen-protein adducts were detected in the serum of only 1 study subject, a patient with marked transaminase elevation (> 6,000 IU/L) and high risk for the development of hepatotoxicity according to the Rumack nomogram. While this study provides further support for the occurrence of covalent binding of acetaminophen to hepatic protein in humans following acetaminophen overdose, the detection of acetaminophen-protein adducts in serum with the current methodology requires significant biochemical evidence of hepatocellular injury.

Evaluation of occult acetaminophen hepatotoxicity in hospitalized children receiving acetaminophen. Pediatric Pharmacology Research Unit Network.

The safety of repeated doses of acetaminophen in ill children with the potential of reduced glutathione stores has been questioned. This study measured hepatic transaminases in children and adolescents (n=100) who received > or = 6 therapeutic doses of acetaminophen over a 48-hour period of hospitalization. Acetaminophen-protein adducts were measured in a cohort of subjects with hepatic transaminase elevation (n=8) and in those (n=10) receiving concurrent drug therapy with agents that induce the cytochrome P450 enzymes involved in acetaminophen metabolism. Acetaminophen-protein adducts were not detected in this cohort of 18 subjects. Based on this pilot study, the routine use of acetaminophen at therapeutic doses in ill, hospitalized children and adolescents appears safe.

CD4(+) T-cell activation and induction of autoimmune hepatitis following trichloroethylene treatment in MRL+/+ mice.

Exposure to relatively high levels of trichloroethylene has recently been shown to accelerate the development of an autoimmune response in the autoimmune prone MRL+/+ mice. The trichloroethylene-induced autoimmune response was associated with an increase in activated CD4(+) T cells, producing Th(1)-like cytokines. The present study was conducted to determine whether lower, more occupationally relevant doses of trichloroethylene could also promote autoimmunity, in MRL+/+ mice, and if so, to investigate the mechanism of this accelerated autoimmune response. In addition, histological studies were performed to determine if trichloroethylene was capable of producing pathological markers consistent with an autoimmune disease. Trichloroethylene was administered to mice in the drinking water at 0, 0.1, 0.5, and 2.5 mg/ml for 4 and 32 weeks. There was a significant increase above controls in serum antinuclear antibody (ANA) levels following 4 weeks of both 0.1 and 0.5 mg/kg/day of trichloroethylene. After 32 weeks of treatment, ANA levels were elevated and equal in all groups. The kinetics of the ANA response indicated that trichloroethylene accelerated the innate autoimmune response in the MRL+/+ mice. There was a dose-related increase in the percentage of activated CD4(+) T cells in both the spleens and lymph nodes of mice treated for 32 weeks with trichloroethylene when compared to controls. CD4(+) T cells isolated from MRL+/+ mice after either 4 or 32 weeks of treatment with trichloroethylene secreted inflammatory or Th(1)-like cytokines. Following 32 weeks of trichloroethylene treatment, there was a significant increase in hepatic mononuclear infiltration localized to the portal region, a type of hepatic infiltration consistent with autoimmune hepatitis. Taken collectively, these data suggest that exposure to occupationally relevant concentrations of trichloroethylene can accelerate an autoimmune response and can lead to autoimmune disease. The mechanism of this autoimmunity appears to involve, at least in part, activated CD4(+) T cells that then produced inflammatory cytokines.

Inhibition of CYP2E1 reverses CD4+ T-cell alterations in trichloroethylene-treated MRL+/+ mice.

Trichloroethylene is an organic solvent that is primarily used as a degreasing agent for metals. There is increasing evidence in both humans and animal models that trichloroethylene promotes the development of autoimmunity, but little is known about the mechanisms that mediate the effect of trichloroethylene on the immune system. Metabolic activation of trichloroethylene is considered an obligatory pathway for other known toxicities such as hepatotoxicity, nephrotoxicity, and carcinogenicity. Trichloroethylene is metabolized by the cytochromes P450, primarily cytochrome P450 2E1 (CYP2E1). To investigate whether metabolism by CYP2E1 is required for immunomodulation, we treated autoimmune prone MRL+/+ mice with trichloroethylene in the drinking water for 4 weeks, in the presence or absence of diallyl sulfide, a specific inhibitor of CYP2E1. Using an antibody that recognizes proteins covalently modified by a reactive metabolite of trichloroethylene; two immunoreactive proteins were detected in liver microsomes from trichloroethylene-treated mice. Formation of these trichloroethylene-protein adducts, an indicator of metabolic activation, was completely inhibited in animals that were concomitantly treated with trichloroethylene and diallyl sulfide. The level of CYP2E1 apoprotein in liver microsomes was significantly reduced in the presence of diallyl sulfide. The enhanced mitogen-induced proliferative capacity of T cells from trichloroethylene-treated MRL+/+ mice was inhibited if the mice were also treated with diallyl sulfide. In addition, the reduction in interleukin-4 levels secreted by activated CD4+ T cells from trichloroethylene-treated mice was reversed if the mice were also treated with diallyl sulfide. Taken collectively, metabolism of trichloroethylene by CYP2E1 is responsible, at least in part, for the CD4+ T cell alterations associated with exposure to this environmental toxicant.

Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice.

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.