ABSTRACT. Pseudomonas aeruginosa Exotoxin A (PEA) has been generally used to induce liver injury in mice for experimental study. No PEA-induced hepatotoxicity study has ever been conducted in rats, although rats are the most common rodents used in toxicologic bioassay and pharmacological evaluation. The present study was conducted in male Wistar rats that were injected (i.v.) with PEA at doses of 0, 10, 20, 30 or 40 µg/kg body weight and evaluated at 12, 24, 36, 48 and 60 hr post-exposure (HPE). Rats exposed to PEA at 40 µg/kg died before 36 HPE, and the mortality was dose and time dependent. Liver injury was noted as increases in serum enzymes, along with alterations of liver histology in the 40 µg/kg group at 12 HPE. TUNEL-positive staining indicative of hepatocyte apoptosis was observed in the 20 µg/kg group at 12 HPE. Significant levels of DNA fragmentation ladder were observed in the 30 µg/kg group starting at 24 HPE. Serum levels of TNF-α was increased in the 30 and 40 µg/kg groups at 48 and 24 HPE, respectively. Other cytokines, such as IL-2, IL-6, and IL-10 were also increased at various doses and times. Furthermore, the elevated serum heaptic index levels decreased significantly by dexamethasone pretreatment. In contrast, these markers were exacerbated by co-administration of a non-toxic dose LPS. In overall evaluation, the PEA-induced liver injury can be used as a model for study of hyperimmune-mediated hepatotoxicity.

These results suggested that Kupffer cells, TNF-α and TLR-4 play central mediator roles during the hepatoprotection against PEA-induced hepatotoxicity conferred by LPS.

ABSTRACT. Epidemiological investigations suggest that increased age is associated with susceptibility to infection. Pseudomonas aeruginosa (P. aeruginosa) infection and associated exotoxin A (PEA) toxicity have been reported in hospitalized elderly patients and young children with cystic fibrosis. The present study investigated age-related differences in PEA-induced hepatotoxicity in post weaning (PW, 3 weeks), young adult (YA, 12 weeks), and mature adult (MA, 60-64 weeks) rats. PEA (20 µg/kg) was injected intraveneously and mortality, clinical chemistry, hepatic histopathology, TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling) and PCNA (Proliferating cell nuclear antigen) staining, and serum cytokine levels were assessed at specific time points, up to 72 hr post-exposure (HPE). Mortality in MA rats was 100% at less than 48 HPE. Serum ALT levels in MA rats were approximately 5-fold greater than levels in PW and YA rats at 36 HPE. MA rat liver histological sections showed diffuse hepatocellular necrosis. In contrast, hepatocellular apoptosis, demonstrable by the TUNEL method, was noted simply in the periportal and midzonal regions from 36 to 48 HPE. Increased morphological mitoses and PCNA-positive hepatocytes were seen in PW and YA rats at 72 HPE. These parameters were correlated with age-dependent significant increases in TNF-α, IL-2, IL-6, and IL-18 levels. These data suggest that inflammatory cytokines play an important role in age-related differences in PEA-induced hepatotoxicity. Moreover, these cytokines might correlate with different patterns histopathologic features at various ages.

Pseudomonas aeruginosa Exotoxin A (PEA) induces hepatotoxicity in experimental animals. Lipopolysaccharide (LPS) interacts synergistically with xenotoxics to induce severe organ injury. We examined the combination of non-injurious doses of LPS and sub-hepatotoxic PEA in the induction of multiple organ injury (MOI). Rats treated with 20 or 40 microg/kg LPS plus 10 microg/kg PEA developed severe liver, kidney, and lung injury; elevation of TNF-alpha, IFN-gamma, and IL-2; and high mortality. Depletion of Kupffer cells or T-cells by pretreatment with Gadolinium Chloride or FK506, respectively, attenuated MOI. Thus LPS + PEA acted synergistically on Kupffer and T-cells to induce proinflammatory cytokines contributing to MOI.

The present study investigated the effect of different bacterial species lipopolysaccharide plus Pseudomonas exotoxin A (LPS/PEA) on the induction of multiple organ injury (MOI). Rats were injected with various LPS from Salmonella (SAE, SAT), E. coli (EB4, EB5), or P. aeruginosa (PAL) and PEA showed a greater mortality in the SAE/PEA and SAT/PEA groups. Histological alterations, serum enzymes, and cytokines changes were severer in the SAE/PEA group than the EB4/PEA or PAL/PEA group. EB4/PEA and PAL/PEA failed to induce MOI, even at the LPS doses increased up to 2-4- and 4-8-fold, respectively. Rats co-treated with Salmonella lipid A/PEA developed severer MOI than the E. coli lipid A/PEA. The results indicated the critical roles of MOI induction, which were related to LPS derived from appropriate bacterial species.