Nitric oxide rescues thalidomide mediated teratogenicity

Abstract

Thalidomide, a sedative drug given to pregnant women, unfortunately caused limb deformities in thousands of babies. Recently the drug was revived because of its therapeutic potential; however the search is still ongoing for an antidote against thalidomide induced limb deformities. In the current study we found that nitric oxide (NO) rescues thalidomide affected chick (Gallus gallus) and zebrafish (Danio rerio) embryos. This study confirms that NO reduced the number of thalidomide mediated limb deformities by 94% and 80% in chick and zebrafish embryos respectively. NO prevents limb deformities by promoting angiogenesis, reducing oxidative stress and inactivating caspase-3 dependent apoptosis. We conclude that NO secures angiogenesis in the thalidomide treated embryos to protect them from deformities.

Figure 1. Nitric oxide recovers the thalidomide teratogenic effects.

(A) Chicken embryos (n = 160 eggs) were treated with 40 μg of thalidomide at HH stage 8 followed by treatment with various concentrations of spNO (0.001 μM – 100 μM). An addition of 10 μM spNO after 30 min of thalidomide treatment could neutralizes the thalidomide teratogenic effect up to 94 %. **p<0.01. (B) In another set of experiments, embryos were treated with 40 μg of thalidomide followed by addition of spNO at different times (0, 0.5, 1, 6, 12, 24 and 144 h) after adding thalidomide. **p<0.05 vs vehicle control; #p<0.01 vs 1 μM.

Figure 2. Nitric oxide mediated recovery is not species specific.

White leghorn (WL), Brown leghorn (BL) (HH 8) and Zebrafish (ZF) embryos (10 hpf) were treated with 40 μg thalidomide in case of chick embryos (n = 50 eggs) or 2 mg/ml thalidomide in case of zebrafish embryos (n = 20 fishes). SpNO (10 μM) was added after 30 min as described previously. In case of zebrafish spNO (10 μM) was added along with thalidomide in the water. Analysis of white and brown leghorn embryos at HH32 and zebrafish embryos at 72 hpf showed limb deformities and pectoral fin deformities respectively. In case of zebrafish (n = 20 fishes), the deformities were scored as presence or absence of the pectoral fins. (A) Plates are representative of whole embryos with limb deformities in thalidomide, spNO and thal+spNO. (A–D) represents deformities in the number of digits in the presence or absence of treatments. (E–H) represents wing deformities as visualized using a cartilage specific, alcian blue stain. (B) Representative images of treated or untreated zebrafish embryos after 72 hpf stained with alcian blue. The arrows indicate the presence or absence of pectoral fins in the zebrafish embryo. (C) Bar graphs represent the percentage of limb deformities present in control, thal, spNO and Thal+spNO treated White Leghorn (WL), Brown Leghorn (BL) and Zebra Fish (ZF) embryos. *p <0.01 vs control; #p< 0.01 vs Thal.

Figure 3. Nitric oxide overexpression rescues thalidomide affected embryos.

(A–D) Zebrafish eggs (2 cell stages) were electroporated with eNOS GFP plasmid or S1179D. The live embryos (10 hpf) (n = 20 fishes) were grown in presence and absence of 2 mg/ml thalidomide containing water. After 80 hpf the GFP expression was observed under the fluorescence microscope. The pectoral fins abnormalities are presented in scrambled vector control, GFP alone and eNOS GFP electroporated fishes (E–H). Zebrafish eggs electroporated with S1179D were treated with NO specific dye, DAR-4M-AM. The NO production in thalidomide treated or untreated fishes were visualized with the fluorescence microscope. Arrows indicate the pectoral fins. (I) The graphs represent relative intensities of GFP expression and NO production calculated from the images using histogram analysis of image J software. NTF = Non transfected fish; TF = Transfected fish; Thal = Thalidomide treated fish; Thal+Tf = Thalidomide treated transfected fish. *p<0.01 vs eNOS GFP; #p = 0.01 vs S1179D. Inset shows eNOS S1179D expression in transfected zebrafish embryos confirmed using reverse transcriptase PCR. (J) 2 cell stage zebrafish eggs were treated with eNOS inhibitor, L-NAME (10 μM). After 72 hpf the fish embryos were scored for pectoral fin abnormalities. *p<0.05 vs Thal.

Figure 4. Nitric oxide recovers angiogenesis.

(A) Representative bright field and DAR images of pre- and post-treated aortic rings. (B) The bright field images were used to calculate the number of tubes after various treatments (n = 5). *p>0.01 vs control; # p>0.01 vs Thal. (C) Fluorescence intensity of aortic rings calculated following incubation with DAR-4M-AM. *p>0.01 vs control; # p>0.01 vs Thal. (D) Embryos were treated with 40 μg thalidomide followed by treatment with spNO (10 μM). Total RNA was harvested from HH17-18 embryo and evaluated for angiogenic genes expression profile. cDNA was primed with avian specific VEGFR2 and Ang1 primers. As an internal control, β-actin mRNA was measured in parallel. Expression of VEGFR2 and Ang1 was analyzed for all treatments. Densitometric analysis of blots relative to β-actin expression. *p <0.05 vs control; #p<0.01 vs Thal.

Figure 5. Nitric oxide inhibits thalidomide induced caspase-3 dependant apoptosis.

(A–C) Endothelial cells or limb cells or chick aorta or limb buds dissected from a 6^th day old (HH17-18) embryo were treated with thalidomide (40 μM) for 4 h spNO (10 μM) was added 5 min after thalidomide treatment. Annexin V and propidium iodide was used to check apoptosis. Apoptotic cells per field (n = 100 fields) were counted from the images taken using a fluorescence microscope. *p<0.05 vs control; #p<0.01 vs Thal. (D) Migration of the limb cells was assayed using scrape wound healing assay. Confluent limb cells were treated with caspase-3 inhibitor DEVD (10 nM) or thalidomide or spNO. A wound was created using a sterile pipette tip and difference in area of the wound was measured before and after 4 h. *p = 0.029 vs Thal; *p<0.01.

Figure 6. Nitric oxide restricted the production of thalidomide induced reactive oxygen species (ROS).

Endothelial cells were treated as described before. Total RNA was by isolated from the cells and reverse transcribed to cDNA using reverse transcriptase enzyme cDNA was amplified with sense and anti sense primers of caspase-3, FOXO1, FOXO3a and GAPDH. (B–D) Eahy926 cells or chick aorta were pre-treated and post treated with thalidomide, spNO or the combination for 4 h. Reactive oxygen species such as hydrogen peroxide, superoxide and peroxynitrite levels were estimated using amplex red, nitroblue tetrazolium and HPF assays respectively. Each of the assays were performed independently and normalized with respect to control values. *p<0.01 vs control; #p<0.01 vs Thal.

Figure 7. Catalase protects avian and zebrafish embryos from thalidomide teratogenicity.

(A) Chick limb buds from chick embryo (n = 50 eggs) were removed at stage HH17-18 aseptically and treated with thalidomide or spNO or catalase (30 Units/ml) for 4 h. The limb buds were then stained with Annexin V and propidium iodide to check apoptosis. *p<0.01vs Thal. (B) 10 hpf zebrafish (n = 20 fish embryos) embryos were treated with thalidomide (2 mg/ml) or catalase (30 Units/ml) up to 72 hpf. #p = 0.0037 vs Thal.