Hemoadsorption Improves Survival of Rats Exposed to an Acutely Lethal Dose of Aflatoxin B1

Mycotoxins, such as aflatoxin B1 (AFB1), pose a serious threat as biological weapons due to their high toxicity, environmental stability, easy accessibility and lack of effective therapeutics. This study investigated if blood purification therapy with CytoSorb (CS) porous polymer beads could improve survival after a lethal aflatoxin dose (LD90). The effective treatment window and potential therapeutic mechanisms were also investigated. Sprague Dawley rats received a lethal dose of AFB1 (0.5–1.0 mg/kg) intravenously and hemoperfusion with a CS or Control device was initiated immediately, or after 30, 90, or 240-minute delays and conducted for 4 hours. The CS device removes AFB1 from circulation and significantly improves survival when initiated within 90 minutes of toxin administration. Treated subjects exhibited improved liver morphology and health scores. Changes in the levels of cytokines, leukocytes and platelets indicate a moderately-severe inflammatory response to acute toxin exposure. Quantitative proteomic analysis showed significant changes in the level of a broad spectrum of plasma proteins including serine protease/endopeptidase inhibitors, coagulation factors, complement proteins, carbonic anhydrases, and redox enzymes that ostensibly contribute to the therapeutic effect. Together, these results suggest that hemoadsorption with CS could be a viable countermeasure against acute mycotoxin exposure.

cells/µL in the Control and CS-treated group, respectively (Table S3). At 8 hours, WBC numbers had rebounded to baseline levels. WBC counts continued to rise through 24 hours in both groups to 13 x10 3 cells/µL in the Control group and 17 x10 3 cells/µL in the CS group and were significantly different from that of baseline (Paired t-test P=0.034 and P=0.045, respectively). However, there was no significant difference between the two groups at this time (P=0.18). Platelet numbers decreased over 8 hours by 23% and 14% for the Control and CS-treated groups, respectively. After 24 hours, platelet counts continued to decline to 73% and 64% of baseline in the Control and CS-treated group, respectively. There was no significant difference between the two groups at this time (P=0.13). Erythrocyte number changed modestly after 24 hours, with levels in the CS-treated group significantly lower than those of the Control group (7.3 x10 6 cells/µL vs. 8.0 x10 6 cells/µL) (P=0.01) (Table S3).

Cytokine Response
Pro-inflammatory cytokines were marginally elevated during the hemoperfusion period in the immediate treatment animals, with no significant differences between Control and CS-treated groups ( Figure S1 and Table S4). AFB1 caused a delayed inflammatory effect with substantial increases in pro-inflammatory cytokines IL-1α, IL-1β, IL-2, IL-5, IL-6, IL-12, TNF-α, and IFN-γ d above baseline for both study groups at 24 hours. After 2 days, all eight cytokines were elevated, with the CS-treated group exhibiting higher overall levels of IL-5, IL-6, IL-12, TNF-α, and IFN-γ than the remaining Control animals, despite the single CS treatment occurring immediately after toxin exposure ( Figure S1 and Table S4). Anti-inflammatory cytokines IL-4, IL-13, and GM-CSF had a modest increase in the CS-treated rats in the days following the AFB1 dose, with IL-4 and IL-13 peaking on day 2 at 73 and 101 pg/mL, respectively, and GM-CSF peaking on day 3 at 381 pg/mL ( Figure S1 and Table S4). Control rats experienced a similar increase for IL-4. IL-13 and GM-CSF peaked at lower levels in the Control rats, but these differences were not significant. IL-10, also an anti-inflammatory cytokine spiked just 1.5 hours after toxin insult in both CS-treated and Control groups. IL-10 levels in the CS group were blunted in comparison to those of the Control group: 198 pg/mL vs. 550 pg/mL; however, this difference was not significant (t-test P=0.13). The following days, IL-10 levels dropped in the Control group, whereas IL-10 dropped briefly in the CS-treated group, before increasing to a peak level of 351 pg/mL on day 6. By day 7, all cytokine levels in the CS-treated group had declined considerably, with IL-6, IL-13, TNF-α, and IFN-γ returning to baseline levels ( Figure S1 and Table S4).
In the 30-minute delayed treatment study, cytokine levels gradually rose in both Control and CStreated groups for the first 4 days after dosing and were almost identical for IL-1α, IL-2, IL-4, IL-5, IL-10, and IFN-γ ( Figure S2 and Table S5). The CS-treated group experienced a smaller spike at 4.5 hours to 204 pg/mL before dropping back down to 120 pg/mL at 1 day. However, this difference was not significant (t-test P=0.33). IL-1β levels increased slightly in Control group rats over the 4 days to 179 pg/mL, whereas CS-treated rats experienced a larger increase to 6345 pg/mL; though this difference was not significant (t-test P=0.31) ( Figure S2 and Table S5). TNFα and IL-6 levels peaked sharply at 1.5 and 4.5 hours, respectively for both Control and CS-treated groups. After day 4, only CS-treated rats survived and continued to experience an increase in cytokines through day 6 before declining on day 7 ( Figure S2 and Table S5).
In the 90-minute delayed treatment study, there was a sharp decline in all tested cytokine levels immediately after toxin dosing which persisted through 5.5 hours. Afterwards, cytokines increased on day 1, peaking between day 2 and 3 in both Control and CS groups ( Figure S3 and Table S6). There were no differences in cytokine levels between the two groups throughout the 7-day observation period. For GM-CSF and IL-1β, the CS group experienced a more rapid reduction in these cytokine levels after day 4 than observed in Control animals, although these differences were not significant ( Figure S3 and Table S6).
During the 4-hour delayed treatment study, all tested cytokines decreased to levels below those of baseline at 4 hours post-toxin dose and began to increase in concentration by 8 hours ( Figure S4 and Table S7). Cytokine levels continued to increase on day 1, with the majority peaking on day 2 through 7 in both Control and CS-treated groups. Cytokine levels did not differ significantly between the Control and CS-treated during the 7-day study.  Table S10. Effect of CS treatment on AFB1 (1mg/kg)-induced changes in circulating protein abundance following 4-hr hemoperfusion (n=8). All listed proteins were significantly impacted (P<0.05). Change in levels is calculated as fold change in abundance (CS/Control at 5.5h post toxin dose).