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

Abstract

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.

Figure 1

Circulating AFB₁ Levels During Hemoperfusion. AFB₁ levels measured by ELISA from plasma collected during hemoperfusion immediately after AFB₁ IV administration (0.5 mg/kg). Hemoperfusion used either an empty (Control) or a CS device. Mean ± SD. n = 4 for both groups, *P < 0.05.

Figure 2

Survival Plots. Effect of hemoperfusion on survival following a lethal AFB₁ dose. Rats were dosed with AFB₁ IV and subjected to hemoperfusion with a Control or a CS device for 4 hours either (A) immediately after toxin injection (0.5 mg/kg AFB₁); n = 8 and 10 for Control and CS groups, respectively, *P < 0.025, (B) after a 30-min delay (0.5 mg/kg AFB₁); n = 8 for both groups, *P < 0.003, (C) after a 90-min delay (0.5 mg/kg AFB₁); n = 12 and 11 for Control and CS groups, respectively, *P < 0.04, or (D) after a 4-h delay (1 mg/kg AFB₁); n = 8 for both groups.

Figure 3

Body Weights. AFB₁ -induced body weight loss. Rats received 0.5 mg/kg AFB₁ dose (IV) (immediate, 30-min, and 90-min delayed treatment) or 1 mg/kg dose (4-h delayed treatment) and after the indicated delay, were connected to a hemoperfusion circuit containing either a Control or a CS device. Mean ± SD, T0 n ≥ 8. Dashed line represents sole surviving control rat, *P < 0.05. Red line indicates threshold for euthanasia.

Figure 4

Liver Pathology. Liver pathology caused by acute AFB₁ intoxication. H&E stained liver sections from rats injected with AFB₁ IV and treated with either a Control or CS device as indicated. Areas of severe hemorrhage and hepatocyte necrosis are visible in tissues at day 1. (A–D) On day 3–4, leukocyte infiltration is present. (G,H) Biliary hyperplasia is evident in the day 7 toxin-exposed animals (F,I,J). 100X mag.

Figure 5

Histological Scores. Scores of liver sections from AFB₁ injected animals. Treatment with either a Control or CS device began immediately (A), after a 30-min delay (B), a 90-min delay (C), or a 4-h delay. (D) Dose: 0.5 mg/kg AFB₁ for immediate, 30-min, and 90-min delayed treatments; 1 mg/kg for 4-h delayed treatment. Key markers of toxin-induced damage: necrosis, hemorrhage, inflammation, and hyperplasia were scored on increasing severity from 0 through 4, with 0 representing normal healthy tissue.

Figure 6

Hepatocyte Apoptosis. AFB₁-induced hepatocyte apoptosis in rats treated for 4 hours with either a Control or CS device 90-minutes after AFB₁ dosing (1.0 mg/kg) then sacrificed. Liver sections were TUNEL stained to visualize apoptotic cells (stained brown) (A,B) or H&E-stained for gross morphology (C,D). 400X mag.

Figure 7

Cytokine Levels. Effect of AFB₁ injection and subsequent hemoperfusion on levels of key circulating pro-inflammatory cytokines. AFB₁ dose (mg/kg) was administered at time 0 with the hemoperfusion starting immediately (0.5 mg/kg), at 30 min (0.5 mg/kg), at 90 min (0.5 mg/kg), or at 4 hours (1.0 mg/kg). Mean ± SEM.

Figure 8

Proteomic Analysis. Effect of CS treatment on AFB₁ (1.0 mg/kg)-induced changes in circulating protein abundance at 5.5 hours (n = 8). Key proteins that were significantly different between Control and CS treatment (P < 0.05) are color coded by function: red, hemoglobin; black, pH and fluid homeostasis; orange, coagulation; purple, complement; green, protease inhibitor; blue, ROS detoxification; brown, heat shock. Mean difference (Control-CS) vs. statistical P-value at 5.5 h post-toxin dose following 4 hours of hemoperfusion.