Toxicity profiles and protective effects of antifreeze proteins from insect in mammalian models

Abstract

Antifreeze proteins (AFPs), found in many cold-adapted organisms, can protect them from cold and freezing damages and have thus been considered as additional protectants in current cold tissue preservation solutions that generally include electrolytes, osmotic agents, colloids and antioxidants, to reduce the loss of tissue viability associated with cold-preservation. Due to the lack of toxicity profile studies on AFPs, their inclusion in cold preservation solutions has been a trial-and-error process limiting the development of AFPs' application in cold preservation. To assess the feasibility of translating the technology of AFPs for mammalian cell cold or cryopreservation, we determined the toxicity profile of two highly active beetle AFPs, DAFP1 and TmAFP, from Dendroides canadensis and Tenebrio molitor in this study. Toxicity was examined on a panel of representative mammalian cell lines including testicular spermatogonial stem cells and Leydig cells, macrophages, and hepatocytes. Treatments with DAFP1 and TmAFP at up to 500 μg/mL for 48 and 72 h were safe in three of the cell lines, except for a 20% decrease in spermatogonia treated with TmAFP. However, both AFPs at 500 μg/mL or below reduced hepatocyte viability by 20-40% at 48 and 72 h. At 1000 μg/mL, DAFP1 and TmAFP reduced viability in most cell lines. While spermatogonia and Leydig cell functions were not affected by 1000 μg/mL DAFP1, this treatment induced inflammatory responses in macrophages. Adding 1000 μg/mL DAFP1 to rat kidneys stored at 4 °C for 48 h protected the tissues from cold-related damage, based on tissue morphology and gene and protein expression of two markers of kidney function. However, DAFP1 and TmAFP did not prevent the adverse effects of cold on kidneys over 72 h. Overall, DAFP1 is less toxic at high dose than TmAFP, and has potential for use in tissue preservation at doses up to 500 μg/mL. However, careful consideration must be taken due to the proinflammatory potential of DAFP1 on macrophages at higher doses and the heighten susceptibility of hepatocytes to both AFPs.

Keywords: Antifreeze proteins; Cell lines; Cold protection; DAFP1; Kidney; Toxicity.

Figure 1.. Effect on AFPs on the viability of testicular C18-4 spermatogonia and MA-10 Leydig cells.

(A-B) Effect of 0 to 2000 μg/mL DAFP1 on C18-4 cell viability measured by MTT assay over (A) 48h and (B) 72h treatments. (C-D) Changes in viability with TmAFP treatments at 0 to 500 μg/mL over (C) 48h and (D) 72h. (E-G) Effect of 0 to 1000 μg/mL DAFP1 on MA-10 cell viability measured by MTT assay over (E) 48h and (F) 72h treatments. (G) MA-10 cell proliferation measured by EDU incorporation assay in control cells (0) and cells treated with 1000 μg/mL DAFP1 for 48h. (H-I) Changes in MA-10 cell viability with TmAFP treatments at 0 to 1000 μg/mL over (H) 48h and (I) 72h. Results are presented as fold change of vehicle control. N=3 independent experiments conducted in triplicates. Significant difference relative to vehicle control with One way ANOVA test and multiple comparisons: * (p≤0.05), ** (p<0.01), *** (p<0.001).

Figure 2.. Effect on AFPs on RAW 264.7 macrophage viability.

(A-B) Effect of vehicle (0) and up to 1000 μg/mL DAFP1 on RAW 264.7 macrophage viability measured by MTT assay over (A) 48h and (B) 72h treatments. (C-D) Effect of vehicle (0) and TmAFP at up to 1000 μg/mL on RAW 264.7 cell viability measured by MTT assay over (C) 48h and (D) 72h treatments. Results are presented as fold change of vehicle control. N=3 independent experiments conducted in triplicates. Significant difference relative to vehicle control with One way ANOVA test and multiple comparisons: * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 3.. Effect of AFPs on HUH7 hepatocyte Viability and proliferation.

Cell viability was determined in HUH7 cells treated with vehicle (0) or up to 1000 μg/mL of DAFP1 (A-D) or TmAFP (E-F) for 48h or 72h by MTT assay. Proliferation was measured by EDU incorporation assay, with representative images of EDU incorporation shown in C (scale in μm.), and quantification shown in D. Results represent the means ± SEM of 3 independent experiments conducted each in triplicates and are presented as fold change of vehicle control (0 μg/mL). Significant differences relative to vehicle control were calculated with One way ANOVA test and multiple comparisons or t-test: * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 4.. Effect of DAFP1 on germ cell markers in C18-4 Spermatogonia.

Effect of 48h treatments with vehicle or 1000 μg/mL DAFP1 on select spermatogonial markers (A) Id4 (B) Foxo1 and (C) Mcam, and on spermatocyte marker Sycp1 (D). Results are presented as fold change of vehicle control. qPCR data are normalized to GAPDH. N=3 independent experiments conducted in triplicates. Significant difference relative to vehicle control with t-test: * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 5.. Effect of DAFP1 on MA-10 Leydig cell Progesterone Synthesis.

Effect of 48h treatments with vehicle or 1000 μg/mL DAFP1 on MA-10 cells progesterone production in the presence or absence of 50 ng/mL hCG added for 2h at the end of treatment. Results are normalized to total protein. N=3 independent experiments conducted in triplicates. Significant difference relative to vehicle control with t-test: * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 6.. Effect of DAFP1 on the expression of inflammatory genes and TNFa cytokine in RAW 264.7 macrophages.

Effect of 40h treatment with vehicle or 1000 μg/mL DAFP1 on (A-D) gene expression of the inflammatory genes Il1b, Cxcl2, Tnfa, and Cxcl10 measured by qPCR analysis, normalized to HPRT. (E) Effect of 40h treatment with vehicle or 1000 μg/mL DAFP1 on TNFa secreted protein measured by ELISA assay. Results are normalized to total RNA content in cells. N=3 independent experiments conducted in triplicates. Significant difference relative to vehicle control with Student t-test: *** p≤0.001.

Figure 7.. Effect of DAFP1 on the expression of apoptosis-related genes and proliferation marker PCNA in RAW 264.7 macrophages.

Effect of 40h treatment with vehicle or 1000 μg/mL DAFP1 on (A-B) pro-apoptotic genes Bad and Bim, and (C) anti-apoptotic gene Bcl2; and (D) proliferation marker Pcna. qPCR data are normalized to HPRT. N=3 independent experiments conducted in triplicates. Significant difference relative to vehicle control with Student t-test: *** p≤0.001

Figure 8.. DAFP1 internalization in mammalian cell lines.

Representative merge images of bright field and FITC signal in cells illustrating the internalization of FITC-BSA and FITC-DAFP1 into HUH7 cells (A-B) and RAW 264.7 cells (C-D) after 6h of treatment with either medium, 1000 μg/mL of FITC-BSA or FITC-DAFP1. Scales in μm. (E) Quantification of relative fluorescent units (RFU) measured in RAW 264.7 cell lysates. N=2 independent experiments conducted in triplicates. Significant difference relative to FITC-BSA fluorescence using One way ANOVA test and multiple comparisons. * p≤0.05.

Figure 9.. Effect of DAFP1 and TmAFP on the morphology and expression of functional markers in rat kidneys kept under hypothermic conditions.

The expression of gene and protein levels of Aqp1 (Aquaporin water channel 1) and Tcf21 (Pod1) were examined in PND6 rat kidneys kept at 4°C for 48h (A, B, E-G) or 72h (C, D) in the presence of medium (Control/0) or 1000 μg/mL DAFP1 or TmAFP, in comparison to kidneys frozen at the time of dissection (Time zero; T0). The kidneys of 3 rats were used per condition for 48h cold exposure and 2 to 3 rats per condition for 72h cold exposure. After 48h cold treatment, for each rat, one kidney was frozen for mRNA analysis and the other kidney was fixed in paraformaldehyde for immunofluorescence analysis. After 72h cold treatment, all kidneys were processed for qPCR analysis. The mRNA levels of Tcf21 (A, C) and Aqp1 (B, D) were determined by qPCR analysis and normalized to GAPDH. Gene expression in control and DAFP1- or TmAFP- treated tissues kept in cold condition is expressed as fold change of the levels measured in samples frozen after dissection (T0). Statistical significance: * p≤0.05, ** p≤0.01. (E-G) Representative pictures showing the general morphology and immunofluorescence signals of Tcf21 and Aqp1 in kidneys at time zero (E) vs tissues kept at 4°C for 48h with medium (Control) (F) or 1000 μg/mL DAFP1 (G). White arrows point at glomeruli, red arrows point at tubules, and red stars indicate areas of tissue degeneration.

Figure 9.. Effect of DAFP1 and TmAFP on the morphology and expression of functional markers in rat kidneys kept under hypothermic conditions.

The expression of gene and protein levels of Aqp1 (Aquaporin water channel 1) and Tcf21 (Pod1) were examined in PND6 rat kidneys kept at 4°C for 48h (A, B, E-G) or 72h (C, D) in the presence of medium (Control/0) or 1000 μg/mL DAFP1 or TmAFP, in comparison to kidneys frozen at the time of dissection (Time zero; T0). The kidneys of 3 rats were used per condition for 48h cold exposure and 2 to 3 rats per condition for 72h cold exposure. After 48h cold treatment, for each rat, one kidney was frozen for mRNA analysis and the other kidney was fixed in paraformaldehyde for immunofluorescence analysis. After 72h cold treatment, all kidneys were processed for qPCR analysis. The mRNA levels of Tcf21 (A, C) and Aqp1 (B, D) were determined by qPCR analysis and normalized to GAPDH. Gene expression in control and DAFP1- or TmAFP- treated tissues kept in cold condition is expressed as fold change of the levels measured in samples frozen after dissection (T0). Statistical significance: * p≤0.05, ** p≤0.01. (E-G) Representative pictures showing the general morphology and immunofluorescence signals of Tcf21 and Aqp1 in kidneys at time zero (E) vs tissues kept at 4°C for 48h with medium (Control) (F) or 1000 μg/mL DAFP1 (G). White arrows point at glomeruli, red arrows point at tubules, and red stars indicate areas of tissue degeneration.