PrC-210 Protects against Radiation-Induced Hematopoietic and Intestinal Injury in Mice and Reduces Oxidative Stress

Abstract

The development of safe, orally available, and effective prophylactic countermeasures to protect our warfighters is an unmet need because there is no such FDA-approved countermeasure available for use. Th 1-Propanethiol, 3-(methylamino)-2-((methylamino)methyl) (PrC-210), a synthetic small molecule, is a member of a new family of aminothiols designed to reduce toxicity while scavenging reactive oxygen species (ROS). Our study investigated the protective role of a single oral administration of PrC-210 against radiation-induced hematopoietic and intestinal injury in mice. Pre-treatment with PrC-210 significantly improved the survival of mice exposed to a lethal dose of radiation. Our findings indicated that the radioprotective properties of PrC-210 are achieved by accelerating the recovery of the hematopoietic system, stimulating bone marrow progenitor cells, and ameliorating additional biomarkers of hematopoietic injury. PrC-210 pre-treatment reduced intestinal injury in mice exposed to a lethal dose of radiation by restoring jejunal crypts and villi, reducing translocation of bacteria to the spleen, maintaining citrulline levels, and reducing the sepsis marker serum amyloid A (SAA) in serum. Finally, PrC-210 pre-treatment led to a significant reduction (~10 fold) of Nos2 expression (inducible nitric oxide) in the spleen and decreased oxidative stress by enhancing the antioxidant defense system. These data support the further development of PrC-210 to receive approval from the FDA to protect warfighters and first responders from exposure to the harmful effects of ionizing radiation.

Keywords: PrC-210; acute radiation syndrome; antioxidant; hematopoietic radiation injury; prophylactic countermeasure.

Figure 1

Survival of animals administered PrC-210 (450 mg/kg) or water PO 1 h prior to exposure to 9.35 Gy TBI. Animals administered PrC-210 included two cohorts (n = 24/cohort); (i) fasted for 5 h prior to drug administration, (■) and (ii) fed ad libitum (●); survival for both groups was 88%. Percent survival for animals administered the vehicle (included two cohorts (n = 24/cohort)) was 21% in the fasted group (□) and 25% in the fed ad libitum group (○). The log-rank analysis of the water and PrC-210 survival curves had a p-value of <0.0001 and Fisher’s exact test with a p < 0.0001 for both conditions. There were no significant differences between the fasted and fed animals.

Figure 2

Dose optimization of PrC-210. Groups included vehicle (○) or PrC-210 at doses of 250 mg/kg (■), 450 mg/kg (▲), 600 mg/kg (●), and 800 mg/kg (♦). Survival for the 800 mg/kg groups was 63%, the 450 mg/kg group was 54%, the 600 mg/kg group was 46%, the 250 mg/kg was 42%, and the survival in the group administered water was 8%. Log-Rank analyses of all PrC-210 groups compared to water were significant (p = 0.0001–p = 0.0179), and Fisher’s exact test of all PrC-210 groups compared to water was as significant (p = 0.0002–p = 0.0173). There were no significant differences between groups administered PrC-210.

Figure 3

Recovery of peripheral blood cells following radiation injury. (A). white blood cells (WBCs), (B). neutrophils (NEU), (C). platelets (PLT), (D). monocytes (MONO), and (E). lymphocytes (LYM)). Day 0 represents 8 h post-irradiation. Non-irradiated mice treated with water (○) and PrC-210 (●) and irradiated (7 Gy) mice treated with water (□) and PrC-210 (■). Data shown are mean ± standard error of the mean (SEM) for n = 10 mice. Significant differences (p < 0.001–0.0125) between PrC-210-treated and water-treated irradiated groups by ANOVA are indicated with an asterisk (*). Some data points in the figure do not have error bars that are visible because they are smaller than symbols.

Figure 4

Recovery of femoral bone marrow by clonogenic assay. Bone marrow colony-forming units (CFU) were estimated in the femurs of the animals administered either water or PrC-210 (450 mg/kg) 1 h prior to irradiation (7 Gy) and non-irradiated animals (0 Gy) to quantify the recovery. Significant difference *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 5

Recovery of cellularity of sternal bone marrow was determined on days 0 (8 h post-TBI), 1, 3, 7, 14, and 30 post-TBI (7 Gy). The number of megakaryocytes was counted in all four groups (water-treated (0 and 7 Gy) and PrC-210-treated (0 and 7 Gy). The irradiated PrC-210 group showed significant recovery (** p < 0.01) compared to the saline group. Representative H&E-stained sections of sternal bone marrow are shown. Higher loss of cellularity and higher numbers of adipocytes are seen in the irradiated water-treated group compared to the PrC-210-treated group.

Figure 6

Serum levels of FLT3-L (A) and EPO (B) on various days post-TBI (7 Gy) were measured by ELISA. Significantly lower (** p ≤ 0.01) FLT3L levels were observed from day seven onwards in the PrC-210-treated group. In the case of EPO levels, significant recovery (**** p ≤ 0.0001) by PrC-210 treatment was seen on day 14 post-TBI when compared to the water group. Represented data are mean ± standard error of the mean (SEM) for n = 4 mice.

Figure 7

Differential expression levels of genes in the oxidative stress pathway and recovery by PrC-210 assessed by RTPCR. Serum samples from days 1, 7, and 14 post-TBI (7 Gy) were subjected to an array of 89 genes from the oxidative stress pathway. Dotted lines indicate threshold for significant difference. Gene expression analysis on day one showed a 20-fold up-regulation of Nitric oxide synthase 2 (Nos2) in irradiated water-treated mice. Irradiated mice administered PrC-210 significantly downregulated the Nos2 expression. This finding supports a Nos2 role in the radioprotective effects of PrC-210. Nos2 encodes an inducible enzyme that produces nitric oxide, a free radical and major messenger molecule.

Figure 8

Histological evaluation of jejunum on days one, three, seven, and nine post-TBI. (A) H&E-stained representative sections of jejunum from naive, irradiated (9.75 Gy) water, and PrC-210-treated groups. Damage to villi due to radiation was observed by day three. (B) Viable crypts in all three groups were quantitated. Significant recovery in the PrC-210-treated group was observed when compared to the water-treated group on days three and seven by ANOVA (*** p ≤ 0.001, **** p ≤ 0.0001).

Figure 9

Protection of GI tissue by PrC-210. (A) Bacterial translocation of gut bacteria to liver and spleen assessed by PCR. Gut bacteria load in the spleen and liver were estimated in irradiated animals from both groups at both time points. There was a significant difference (t-test * p = 0.017) between the water-treated and PrC-210-treated groups on day one post-TBI in the spleen. (B) H&E-stained representative sections of jejunum from naïve. A significant increase (** p ≤ 0.01) in serum citrulline in the PrC-210 group compared to the control on day three. (C) PrC-210 administration inhibited the radiation-induced elevated synthesis of sepsis marker Serum Amyloid A (SAA) in mouse serum compared to water-treated animals. The levels of SAA were evaluated in serum from samples collected on days one, three, seven, and nine post-TBI by ELISA. Represented data are mean ± standard error of the mean (SEM) for n = 4 mice per group; *** p ≤ 0.001, **** p ≤ 0.0001. Plasma levels of SAA were very high following irradiation. The PrC-210 group showed significantly lower SAA levels compared to the water group on all three days tested.