Animal feeding trial and sample collection

We measured the total cyanide in samples of bamboo shoots and feces and thiocyanate levels in urine in 20 adult healthy giant pandas at the Chengdu Research Base of Giant Panda Breeding (Research Base). During the course of a 28-day feeding trial, bamboo shoots and clean drinking water were provided ad lib. Bamboo shoots used in the trial were from Chimonobambusa szechuanensis and gathered from the habitat of free giant pandas in Dujiangyan, Sichuan province. We recorded intake of bamboo shoots and feces and urine output for each study animal daily. Every 4 days, we sampled 500 g of bamboo shoots chosen casually from the food, about 500 g of feces, and 2 mL of urine from each animal in the morning. Following collection, we immediately measured total cyanide in bamboo shoots and feces, and stored urine samples at −20 °C until analysis.

To determine the rhodanese expression and activity in liver and kidney, we purchased 5 healthy New Zealand white rabbits (10 months old) and 5 healthy domestic cats (1–2 years old) from Animal Experiment Center, Sichuan University. We sacrificed rabbits and cats by cervical dislocation under light ether anesthesia. We quickly excised their liver and kidney and rinsed them in cold physiological saline. We separated organs into 2 portions: we immediately fixed one portion in 10% formol saline for immunohistochemical analysis, and sliced the other portion into thin sections and stored it in liquid nitrogen for real-time RT-PCR and enzyme activity assay.

We collected samples of liver and kidney of adult giant panda from 4 animals that died natural deaths at the Research Base (Stubook numbers 287, 297, 373 and 718) and 1 free giant panda rescued from wild that then died from intestinal obstruction. We also obtained samples from one 9-day-old giant panda that died from trauma at the Research Base. We treated liver and kidney samples of giant pandas as the same as those of rabbits and cats described above.

This study was approved by the Research Base. The methods used were in accordance with the approved guidelines of the institution and followed all regulations of the Research Base. Permission to conduct this research was given by the Director of the Research Base after consultation with the Research, Husbandry and Veterinary Departments. The research protocol and handling procedures were approved by the Directors and staff of the each of those departments.

Measurement of total cyanide

We determined the cyanide level in bamboo shoots or corresponding feces samples according to the method described by Surleva54. We first drew a calibration graph by preparing standard solutions of CN− at concentrations of 0.02, 0.04, 0.08, 0.1.0.2, 0.4 and 0.8 μg/mL by adding appropriate volumes of cyanide solutions at concentration of 20 μg CN−/mL (potassium cyanide dissolved in 0.01 M NaOH) to 1 mL of 2% Na 2 CO 3 . We added 0.5 mL of ninhydrin solution (5 mg/mL ninhydrin in 2% Na 2 CO 3 ) to each standard cyanide solution, homoginized the mixture and incubated it for 15 min for color development. We prepared the blank in the same way as above, except that 1 mL of 2% Na 2 CO 3 without CN− instead of 1 mL 2% Na 2 CO 3 containing CN− was added. We measured UV-Visible absorption of the reaction product (Cyanide-ninhydrin adducts) of the different concentrations of cyanide using an UV/Vis Spectrophotometer (SURGISPEC SM735, Surgical Medical, England) at 485 nm.

We cut samples of bamboo shoots or feces into small sections and ground them in liquid nitrogen. We measured total cyanide in the samples by adding 0.1 g of the ground sample into a standard volumetric flask (5 mL) and making up volume to mark with 0.1% NaHCO 3 . We sonicated samples for 20 min in a water bath and centrifuged the mixture at 10,000 rpm for 10 min. We pipetted the supernatant with an automatic pipette, two aliquots (40 μL each), added 1 mL of 2% Na 2 CO 3 and 0.5 mL ninhydrin solution, allowed 15 min for color development and measured absorbance at 485 nm. We expressed total cyanide content as mg HCN/kg.

Measurement of urinary thiocyanate

We determined thiocyanate levels in giant panda urine by using an improved method described by Lundquist55. Briefly, we applied aliquots of urine (500 μL) diluted with 5.0 mL of 1 M NaOH to columns (2.5 × 0.7 cm) of Amberlyst A-21. We washed the columns 3 times with 5 mL of double-distilled water before eluting thiocyanate by adding 8 mL of 1 M sodium perchlorate. We acidified aliquots of 4 mL of elutes with 0.2 mL of 0.35M acetic acid, and chlorinated for 2 min with 0.1 mL of 50 mM sodium hypochlorite. Then, we added 0.6 mL of color reagent (a mixture of isonicotinic acid and 1, 3-dimethylbarbituric acid) to the treated urinary aliquots. At the same time, we prepared a reagent blank in which urine was replaced with double-distilled water. We performed assays in duplicate. We read thiocyanate concentrations in the samples against a standard curve with known concentrations of potassium thiocyanate. The thiocyanate levels in giant panda urine were expressed as mmol SCN−/L.

Real time RT-PCR

We extracted total RNA using Trizol reagent (Invitrogen, Carlsbad CA, USA). We assessed quality and quantity of the isolated RNA with a Qubit® Fluorometer (Invitrogen, Carlsbad CA, USA) and 1% TBE-Agarose gel electrophoresis, followed by reverse transcription. The 20 μL reverse transcription reaction system comprised the following: 2 μg of total RNA, 1 μL of Oligo (dT) 15 Primer, 10 μL of nuclease-free water, 1 μL of M-MLV Reverse Transcriptase, 1.6 μL of nuclease-free water, 0.4 μL of Recombinant RNase Inhibitor, 4 μL of 5 × reaction buffer, 2 μL of MgCl 2 (25 mM), and 1 μL of PCR Nucleotide Mix. We performed the reaction procedure under the following conditions: denaturation for 5 min at 70 °C, annealing for 5 min at 25 °C, extending annealing for 60 min at 42 °C, inactivated reverse transcriptase for 15 min at 70 °C, and then storage at 4 °C.

We measured mRNA expression level of rhodanese using a 7500 Real-Time PCR System with a 20 μL reaction system containing the following: 1 μL of cDNA, 10 μL of 2 × SYBR Ssofast Evagreen® master mix, 1 μL of each gene-specific primer (100 nM). We performed the reaction procedure as follows: 1 cycle of 95 °C for 30 s, 39 cycles of 95 °C for 5 s, 60 °C for 1 min and 1 cycle of 95 °C for 15 s, 60 °C for 60 s, 95 °C for 30 s, and 60 °C for 15 s. GAPDH was used as a house-keeping gene. We obtained sequences of rhodanese and GAPDH genes for rabbit, giant panda and cat from NCBI database. The primers that we chose from the highly conserved regions for the three species were designed through Oligo 6.0 and Primer 5.0 as rhodanese: 5′-GCGTCGCCCTACGAGATGATG-3′ (forward) and 5′-TTGAGCAGGGAGCGGTCCAG-3′ (reverse) and GAPDH: 5′-TGTCAGCAATGCCTCCTGTA-3′ (forward) and 5′-TTTCCGTTCAGCTCAGGGAT-3′ (reverse), and synthesized by Invitrogen Corporation.

To evaluate the relative quantification of mRNA expression, we normalized the cycle threshold (C T ) values of rhodanese to the C T values of the GAPDH gene and presented results as fold changes of 2 − ∆∆CT. We used the adult giant panda group as control and normalized each transcript level to adult giant panda rhodanese. We calculated relative mRNA expression of the rhodanese in each group using the following equations: ∆C T = C T (Rhodanese) – C T(GAPDH), ∆∆CT = ∆C T(treated group) – ∆C T(control group) .

Immunohistochemistry

We fixed samples of liver and kidney tissues in formalin and embedded them in paraffin. We prepared 5 μm sections, de-paraffinized with Xylene for 10 min and rehydrated in descending ethanol gradient (100–96–70%) solutions. Afterwards, we blocked endogenous peroxidase in the sections for 10 min with 3% H 2 O 2 followed by an antigen retrieval in sodium citrate buffer (pH = 6.0) at 95 °C for 30 min. After 30 min of cooling, we blocked the slides with 10% goat serum (Abcam, UK) and 1% bovine serum albumin (Jackson Immunoresearch, U.S.A.) in PBS for 1 h at room temperature. Then we incubated the sections with 5 μg/mL of a polyclonal rabbit anti-TST primary antibody (Abcam, UK, CAT# ab60128) in a PBS buffer containing 10% goat serum and 1% BSA overnight at 4 °C. After washing four times with PBS, we incubated the sections with 1: 500 of an anti-rabbit antibody conjugated with horseradish-peroxidase (Abcam, UK; CAT# ab6721) for 1 h at room temperature. After a further four washes, we visualized binding sites of the primary antibody with 3, 3′-diaminobenzidine substrate (DAB) (Vector Laboratories, U.S.A.; CAT# SK-4105). Finally we counterstained slides with haematoxyline, dehydrated them in ascending ethanol (70–96–100%) and Xylene, and mounted them with Petrex non-aqueous medium. Negative controls were achieved by the omission of the primary antibody.

We analyzed the degree of rhodanese expressions by a method of integrated optical density (IOD). We excluded scores for the highest and lowest regions for each slide and calculated the average region score from the remaining 10 regions. For comparison purposes, we normalized the IOD value to the entire measured area by calculating IOD/0.25 mm images captured by a relevant software (Olympus DP25), and calculated the IOD sum value using Image-Pro Plus 6.0 software (Media Cybernetics, USA).

Determination of rhodanese activity

We prepared liver and kidney extracts by freezing the samples in liquid nitrogen, homogenizing them with a hand homogenizer, and suspending the homogenates in 25 mM sodium phosphate (pH 7.2). We centrifuged suspensions for 15 min at 4,000 × g, and used supernatants as source of the enzyme. We assayed rhodanese activity by the modified method of Sörbo56. The reaction mixture contained 16.8 mM sodium thiosulphate, 40 mM glycine buffer, pH 9.2, 167 mM KCN and 50 μL enzyme solution in a final volume of 4.0 mL. We carried out the reaction for 15 min at 37 °C and stopped it by adding 0.5 mL 38% formaldehyde. We added formaldehyde in control tubes before the addition of enzyme solution. We determined concentration of thiocyanate as follows: samples were mixed with 1 mL ferric nitrate solution containing 0.025 g Fe(NO 3 ) 3· 9H 2 O in 0.74 mL water and 0.26 mL concentrated nitric acid. We measured absorbance at 460 nm against a blank containing all reagents, except that 50 μL water was used instead of enzyme solution. We obtained concentration of thiocyanate formed from a standard curve produced by treating solutions containing different concentrations of thiocyanate as described above. The unit of enzyme activity was micromole of thiocyanate formed per minute at pH 9.2 and 37 °C. We reported rhodanese activity as specific activity (unit/mg protein) in which total protein was assayed according to Lowry57 using bovine serum albumin as a standard.

Phylogenetic analysis of rhodanese gene

We retrieved the coding sequences of rhodanese genes from GenBank for 11 species including western clawed frog [Xenopus (Silurana) tropicalis (NM_001103038)], human [Homo sapiens (NM_003312)], Norway rat [Rattus norvegicus (NM_012808)], chicken [Gallus gallus (NM_001167731)], house rat [Mus musculus (NM_009437)], polar bear [Ursus maritimus (NW_007907256.1)], amur tiger [Panthera tigris altaica (NW_006712392.1)], domestic cat [Felis catus (NC_018729.2)], giant panda [Ailuropoda melanoleuca (NW_003217342.1)], rabbit [Oryctolagus cuniculus (NC_013672.1)] and sheep [Ovis aries (NC_019460.1)]. After conducting the codon-based multiple alignments, we constructed the Neighbor-Joining (NJ) tree by using the MEGA658, which was out grouped by western clawed frog.

Data analysis

We performed statistical analysis by using the SPSS software package (SPSS Inc., Chicago, IL, USA). We expressed data of feeding trial as mean ± SD and compared female and male giant panda groups by Independent-Sample T Test. We expressed data of rhodanese mRNA and protein expressions and rhodanese activity as mean ± SE and analyzed the data by One-Way ANOVA followed by Dunnet’s test for comparing the results among rabbit, cat and giant panda groups. We showed the newborn panda data as just the one value and a 95% confidence interval value for the adult giant panda data for comparison. P < 0.05 indicates a statistically significant difference.