Interferon (IFN) is a protein which induces the body to produce a broad spectrum of antiviral, antitumour, and immunomodulatory effects after viral infection. It mainly inhibits the growth and reproduction of viruses and exerts antitumour activity by inhibiting viral gene transcription or degrading viral RNA. Interferon-producing cells are divided into three categories I, II, and III according to their biochemical characteristics and the role they play in the body’s immunity. Alpha-type interferon works with the aforementioned broad-spectrum of antiviral effects and exhibits them with high-efficiency
Bovine IFN-α (BoIFN-α) has been proven effective against bovine viral diarrhoea virus (BVDV) (18), foot-and-mouth disease virus (FMDV) (2), and bovine herpesvirus type 1 (12) infections. In addition, there are studies suggesting that recombinant bovine IFN-α has a preventive role in controlling bovine respiratory diseases (17), and that its administration to growing calves could reduce mortality and the incidence of respiratory diseases.
For the reason that IFN is species-specific, research on its toxicology, pharmacokinetics, and general pharmacology is beset by species-specific problems (5). Bovine viral diseases can only be treated with bovine IFN-α and not with human IFN-α. The recombinant human IFN-α drug has been on the market for many years. However, the research on BoIFN-α is still in the laboratory stage, and its development and application in antiviral therapy has yet to gain momentum.
Our group has recently produced recombinant bovine IFN-α (rBoIFN-α) in the
Weight gain (g) = weight after test (g) − weight before test (g);
Weight gain rate (%) = weight gain (g) / weight before test (g) × 100;
Feed utilisation rate (%) = weight gain (g) / feed consumption (g) × 100.
On the 1st day after drug withdrawal (the 31st day of the test) and 1 week later (the 38th day of the test), ten rats were selected from each group, fasted for 12 h, and not given drinking water, and blood was collected retro-orbitally without the bodily sensation for haematology and blood biochemistry examination; after blood collection, the rats were euthanised by cervical dislocation, systematic necropsy and pathological examination were performed, and the observations were recorded.
In the high-dose subgroup, congestion was observed in the lymph nodes of one female rat. Besides, in the high-dose subgroup, there were two other female rats with hyperemia in the ovaries, accompanied by thymic nodules and dark red spots of the thymus being observed in the spleen and thymus. There was also a rough appearance on the surface of the spleen of a male rat in the high-dose subgroup.
Except for the few cases outlined above, compared with the control subgroup, the experimental subgroups showed no significant toxic lesions in the heart, liver, spleen, lungs, adrenal gland, pancreas, intestine, stomach, or ovary. On the first day after drug withdrawal (the 31st day of the test), there was no significant difference between the organ index of rats in each drug dosage subgroup and the blank control subgroup, and they were within the normal physiological range; 1 week after drug withdrawal (the 38th day of the test), the testis index of male rats in the high-dose subgroup was slightly smaller than that of the blank control subgroup (P < 0.05), but there was no significant difference between other organ indexes and the blank control subgroup (Table 1).
Effects of rBoIFN-α on parenchymal organ indexes in Sprague Dawley rats in the sub-chronic toxicity test
Time | Organ | Gender | High-dose group | Middle-dose group | Low-dose group | Control group |
---|---|---|---|---|---|---|
Day 31 | Heart | M | 3.71 ± 0.28 | 3.61 ± 0.28 | 4.02 ± 0.42 | 3.60 ± 0.22 |
F | 3.71 ± 0.19 | 3.88 ± 0.57 | 3.66 ± 0.34 | 4.01 ± 0.43 | ||
Liver | M | 30.01 ± 3.19 | 31.53 ± 3.59 | 31.3 ± 2.28 | 28.14 ± 2.11 | |
F | 33.78 ± 3.10 | 27.46 ± 4.40 | 28.19 ± 1.98 | 32.35 ± 5.27 | ||
Spleen | M | 1.80 ± 0.45 | 2.01 ± 0.40 | 2.01 ± 0.21 | 1.76 ± 0.24 | |
F | 2.01 ± 0.19 | 2.09 ± 0.49 | 2.27 ± 0.99 | 1.89 ± 0.23 | ||
Lung | M | 4.72 ± 0.37 | 5.00 ± 0.54 | 4.61 ± 0.41 | 4.30 ± 1.08 | |
F | 5.54 ± 0.79 | 5.01 ± 0.83 | 5.00 ± 0.24 | 5.82 ± 0.79 | ||
Kidney | M | 7.67 ± 1.24 | 7.53 ± 0.64 | 7.64 ± 0.30 | 7.53 ± 0.70 | |
F | 7.52 ± 0.35 | 7.50 ± 1.54 | 6.35 ± 1.96 | 7.34 ± 0.83 | ||
Stomach | M | 8.74 ± 1.47 | 8.85 ± 1.64 | 9.30 ± 1.39 | 9.30 ± 1.39 | |
F | 9.58 ± 3.20 | 8.44 ± 1.14 | 7.10 ± 1.32 | 7.83 ± 1.73 | ||
Intestines | M | 64.34 ± 11.54 | 64.52 ± 5.79 | 65.48 ± 16.00 | 63.52 ± 3.26 | |
F | 55.22 ± 12.14 | 59.91 ± 11.92 | 58.32 ± 6.33 | 65.08 ± 9.62 | ||
Testis | M | 11.91 ± 0.70 | 12.02 ± 0.62 | 12.26 ± 1.44 | 12.62 ± 1.00 | |
Uterus and ovaries | F | 3.72 ± 1.13 | 4.24 ± 0.73 | 3.88 ± 0.80 | 3.14 ± 0.63 | |
Day 38 | Heart | M | 3.45 ± 0.98 | 3.42 ± 0.21 | 3.35 ± 0.34 | 3.55 ± 0.47 |
F | 3.59 ± 0.53 | 3.60 ± 0.29 | 3.80 ± 0.58 | 3.71 ± 0.45 | ||
Liver | M | 33.01 ± 1.98 | 31.85 ± 2.72 | 32.61 ± 2.83 | 32.06 ± 1.23 | |
F | 27.84 ± 3.20 | 29.50 ± 2.41 | 31.15 ± 2.75 | 29.31 ± 2.26 | ||
Spleen | M | 1.76 ± 0.17 | 1.65 ± 0.17 | 2.01 ± 0.21 | 2.01 ± 0.43 | |
F | 1.83 ± 0.37 | 2.01 ± 0.10 | 2.17 ± 0.91 | 1.80 ± 0.25 | ||
Lung | M | 4.16 ± 0.47 | 4.42 ± 0.57 | 4.25 ± 0.36 | 5.01 ± 1.10 | |
F | 4.27 ± 0.29 | 4.65 ± 0.38 | 4.41 ± 0.24 | 4.64 ± 0.44 | ||
Kidney | M | 6.74 ± 0.30 | 7.04 ± 0.57 | 6.77 ± 0.17 | 7.25 ± 0.36 | |
F | 6.59 ± 0.88 | 6.59 ± 0.58 | 7.03 ± 0.43 | 6.72 ± 0.64 | ||
Stomach | M | 8.66 ± 1.72 | 7.78 ± 0.82 | 8.58 ± 1.45 | 8.53 ± 0.71 | |
F | 8.33 ± 0.86 | 8.07 ± 0.89 | 7.49 ± 1.18 | 7.68 ± 1.74 | ||
Intestines | M | 64.31 ± 8.66 | 66.15 ± 8.74 | 66.88 ± 8.01 | 66.81 ± 7.93 | |
F | 61.07 ± 4.78 | 64.68 ± 11.63 | 58.82 ± 4.52 | 66.23 ± 6.21 | ||
Testis | M | 9.59 ± 0.35* | 10.33 ± 0.70 | 9.75 ± 0.43 | 10.39 ± 0.56 | |
Uterus and ovaries | F | 3.00 ± 0.74 | 2.85 ± 0.19 | 3.32 ± 0.49 | 3.04 ± 0.85 |
* represented significant difference compared with the control group (P < 0.05). M represented male and F represented female
Effects of rBoIFN-α on haematological indexes in Sprague Dawley rats in the sub-chronic toxicity test
Time | Index | Gender | High-dose group | Middle-dose group | Low-dose group | Control group |
---|---|---|---|---|---|---|
Day 31 | HGB (g/L) | M | 151.30 ± 4.16 | 149.00 ± 6.48 | 147.90 ± 4.64 | 146.00 ± 0.11 |
F | 144.10 ± 7.98 | 142.10 ± 8.41 | 142.90 ± 12.65 | 147.50 ± 5.37 | ||
RBC (× 1012/L) | M | 7.72 ± 0.16 | 7.65 ± 0.23 | 7.78 ± 0.33 | 7.63 ± 0.13 | |
F | 7.63 ± 0.39 | 7.49 ± 0.35 | 7.59 ± 0.52 | 7.64 ± 0.37 | ||
WBC (× 109/L) | M | 14.45 ± 1.29 | 13.35 ± 2.26* | 15.07 ± 2.66 | 16.23 ± 1.09 | |
F | 8.54 ± 1.38 | 10.79 ± 1.64 | 8.90 ± 2.31 | 10.11 ± 1.38 | ||
NE (× 109/L) | M | 1.65 ± 0.29 | 1.84 ± 0.90 | 1.86 ± 0.59 | 2.20 ± 0.42 | |
F | 1.18 ± 0.57 | 1.01 ± 0.25 | 1.10 ± 0.64 | 1.41 ± 0.36 | ||
BA (× 109/L) | M | 0.01 ± 0.01 | 0.00 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.00 | |
F | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | ||
EO (× 109/L) | M | 0.10 ± 0.03 | 0.08 ± 0.03 | 0.08 ± 0.04 | 0.10 ± 0.05 | |
F | 0.10 ± 0.03 | 0.10 ± 0.03 | 0.08 ± 0.04 | 0.12 ± 0.05 | ||
LY (× 109/L) | M | 12.31 ± 1.08 | 1.10 ± 2.36 | 12.66 ± 2.40 | 13.01 ± 1.10 | |
F | 7.02 ± 1.13 | 9.56 ± 1.44 | 7.46 ± 1.82 | 8.30 ± 1.30 | ||
MO (× 109/L) | M | 0.26 ± 0.07 | 0.23 ± 0.11 | 0.34 ± 0.11 | 0.33 ± 0.11 | |
F | 0.12 ± 0.08 | 0.10 ± 0.06 | 0.14 ± 0.02 | 0.18 ± 0.05 | ||
Day 38 | HGB (g/L) | M | 145.70 ± 8.87 | 140.50 ± 26.82 | 149.60 ± 5.02 | 155.20 ± 8.38 |
F | 134.50 ± 5.35 | 151.50 ± 9.53 | 143.70 ± 3.49 | 142.70 ± 8.29 | ||
RBC (× 1012/L) | M | 7.39 ± 0.50 | 7.58 ± 0.73 | 7.68 ± 0.58 | 8.06 ± 0.34 | |
F | 7.00 ± 1.27 | 7.66 ± 0.46 | 7.40 ± 0.24 | 7.50 ± 0.28 | ||
WBC (× 109/L) | M | 10.69 ± 3.91 | 10.86 ± 1.50 | 9.01 ± 1.24 | 11.92 ± 4.08 | |
F | 7.42 ± 2.87 | 10.32 ± 1.24* | 8.65 ± 0.92 | 7.33 ± 2.14 | ||
NE (× 109/L) | M | 0.71 ± 0.15 | 1.41 ± 0.54 | 1.05 ± 0.47 | 1.10 ± 0.33 | |
F | 1.00 ± 0.50 | 1.00 ± 0.21 | 0.90 ± 0.33 | 1.00 ± 0.27 | ||
BA (× 109/L) | M | 0.00 ± 0.01 | 0.00 ± 0.01 | 0.00 ± 0.00 | 0.00 ± 0.01 | |
F | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | ||
EO (× 109/L) | M | 0.05 ± 0.03 | 0.11 ± 0.09 | 0.04 ± 0.03 | 0.10 ± 0.05 | |
F | 0.07 ± 0.03 | 0.11 ± 0.04 | 0.10 ± 0.15 | 0.09 ± 0.02 | ||
LY (× 109/L) | M | 9.77 ± 3.84 | 9.03 ± 1.04 | 7.65 ± 0.85 | 10.23 ± 3.69 | |
F | 6.09 ± 2.35 | 8.81 ± 0.83* | 7.32 ± 0.90 | 6.02 ± 2.31 | ||
MO (× 109/L) | M | 0.10 ± 0.05 | 0.18 ± 0.07 | 0.10 ± 0.04 | 0.11 ± 0.06 | |
F | 0.10 ± 0.03* | 0.16 ± 0.05 | 0.11 ± 0.04* | 0.18 ± 0.07 |
HGB: haemoglobin; RBC: red blood cell count; WBC : white blood cell count; NE : neutrophils; BA : basophils; EO: eosinophils; LY: lymphocyte; MO: monocyte. * represented significant difference compared with the control group (P < 0.05). M represented male and F represented female
Effects of rBoIFN-α on blood biochemical indexes in Sprague Dawley rats in the sub-chronic toxicity test
Time | Index | Gender | High-dose group | Middle-dose group | Low-dose group | Control group |
---|---|---|---|---|---|---|
Day 31 | ALT (U/L) | M | 51.10 ± 11.03 | 54.10 ± 7.19 | 46.80 ± 5.87 | 51.40 ± 21.00 |
F | 40.80 ± 7.07 | 43.30 ± 5.08 | 43.60 ± 7.98 | 36.00 ± 11.90 | ||
AST (U/L) | M | 194.70 ± 31.81 | 190.20 ± 54.94 | 175.70 ± 11.80 | 197.30 ± 44.16 | |
F | 203.70 ± 50.69 | 182.80 ± 27.34 | 198.20 ± 29.10 | 188.20 ± 28.74 | ||
BUN (mmol/L) | M | 3.48 ± 0.34 | 4.11 ± 0.84 | 3.56 ± 0.44 | 3.55 ± 0.72 | |
F | 5.00 ± 082 | 5.00 ± 0.65 | 5.30 ± 0.33 | 5.91 ± 1.10 | ||
CREAT (μmol/L) | M | 29.10 ± 3.11 | 29.70 ± 5.63 | 27.50 ± 3.36 | 30.30 ± 1.67 | |
F | 32.70 ± 3.42 | 34.10 ± 4.92 | 33.20 ± 4.83 | 37.10 ± 3.77 | ||
GLU (mmol/L) | M | 3.74 ± 1.34 | 4.02 ± 0.85 | 4.41 ± 0.30 | 4.55 ± 1.58 | |
F | 2.56 ± 0.77 | 2.47 ± 0.24 | 2.90 ± 0.49 | 2.70 ± 0.44 | ||
TP (g/L) | M | 56.84 ± 1.79 | 55.60 ± 3.66 | 56.15 ± 3.89 | 57.10 ± 1.80 | |
F | 61.60 ± 2.33 | 59.72 ± 4.11 | 60.70 ± 3.26 | 60.16 ± 3.54 | ||
ALB (g/L) | M | 42.00 ± 1.07 | 41.88 ± 2.99 | 41.58 ± 2.20 | 41.30 ± 0.48 | |
F | 47.01 ± 1.06 | 45.92 ± 2.80 | 45.58 ± 2.13 | 44.44 ± 2.25 | ||
CHO (mmol/L) | M | 2.03 ± 0.23 | 2.02 ± 0.14 | 2.11 ± 0.20 | 1.81 ± 0.35 | |
F | 1.88 ± 0.36 | 2.00 ± 0.10 | 1.70 ± 0.35 | 1.70 ± 0.30 | ||
TG (mmol/L) | M | 0.74 ± 0.26 | 0.81 ± 0.09 | 1.01 ± 0.14 | 0.64 ± 0.31 | |
F | 0.85 ± 0.29 | 0.79 ± 0.12 | 0.69 ± 0.05 | 0.68 ± 0.19 | ||
Day 38 | ALT (U/L) | M | 49.20 ± 2.30 | 56.20 ± 3.97 | 65.80 ± 15.98 | 60.20 ± 15.47 |
F | 38.50 ± 6.43 | 40.20 ± 5.64 | 42.00 ± 9.88 | 42.70 ± 5.17 | ||
AST (U/L) | M | 125.30 ± 13.07 | 120.20 ± 12.74 | 153.70 ± 28.60 | 143.00 ± 18.15 | |
F | 124.10 ± 24.77 | 121.00 ± 17.03 | 125.50 ± 13.10 | 132.40 ± 13.85 | ||
BUN (mmol/L) | M | 4.73 ± 0.93 | 5.01 ± 0.79 | 4.69 ± 1.06 | 5.10 ± 0.95 | |
F | 6.00 ± 0.32 | 5.77 ± 0.97 | 6.48 ± 0.36 | 5.96 ± 0.66 | ||
CREAT (μmol/L) | M | 29.30 ± 3.91 | 33.10 ± 6.95 | 29.00 ± 4.04 | 31.30 ± 3.91 | |
F | 38.70 ± 4.02 | 38.50 ± 3.65 | 39.20 ± 3.65 | 34.90 ± 2.74 | ||
GLU (mmol/L) | M | 6.40 ± 0.71 | 6.11 ± 0.20 | 5.32 ± 0.79* | 7.10 ± 1.62 | |
F | 4.17 ± 0.74 | 4.70 ± 0.59 | 5.11 ± 0.51 | 4.71 ± 0.70 | ||
TP (g/L) | M | 57.24 ± 2.17 | 58.60 ± 2.46 | 57.16 ± 0.82 | 56.86 ± 1.95 | |
F | 62.44 ± 2.64 | 61.30 ± 4.50 | 63.64 ± 1.34 | 63.02 ± 1.77 | ||
ALB (g/L) | M | 57.24 ± 2.17 | 58.50 ± 2.46 | 57.26 ± 0.82 | 57.00 ± 1.95 | |
F | 31.70 ± 1.55 | 31.80 ± 2.21 | 32.20 ± 2.01 | 32.20 ± 0.79 | ||
CHO (mmol/L) | M | 1.80 ± 0.33 | 1.91 ± 0.05 | 1.63 ± 0.14 | 1.80 ± 0.34 | |
F | 1.80 ± 0.18 | 1.62 ± 0.25 | 1.63 ± 0.10 | 1.77 ± 0.20 | ||
TG (mmol/L) | M | 0.90 ± 0.38 | 1.05 ± 0.52 | 1.02 ± 0.21 | 1.18 ± 0.13 | |
F | 0.91 ± 0.02 | 0.86 ± 0.30 | 0.92 ± 0.06 | 1.11 ± 0.26 |
ALT: alanine aminotransferase; AST : aspartate aminotransferase; BUN : blood urea nitrogen; CREAT: creatinine; GLU: glucose; TP: total protein; ALB: albumin; CHO : cholesterol; TG: triglyceride. * represented significant difference compared with control group (P < 0.05). M represented male and F represented female
Changes in behaviour characteristics: In the acute toxicity and sub-chronic toxicity experiments, the behavioural characteristics of the experimental rats were basically unchanged after injection. Claws and tails were the normal flesh red colour, with no erosion, inflammation, redness, or swelling. Physical activity, scratching ears, flinching feet, and frequent head shaking were not observed. Fur colour was normal and no shedding was observed. Frequent bites of feed were taken and good appetite was noted. Faeces were basically uniform in size, slightly moist, and black. No urinary incontinence was recorded. The animals were responsive to touch.
Deaths: In the acute toxicity experiment, none of the rats died. In the subchronic toxicity experiment, the rats also all lived until the end of experiment.
Changes in body weight: The results showed that in the acute toxicity test, the weight change was relatively smooth, with a slight upward trend, but the difference was not significant. In the subchronic toxicity test, its change also showed an upward trend, and there were no large fluctuations.
Changes in body temperature: After administration, the body temperature changes of the animals in each group were similar in the acute toxicity and subchronic toxicity experiments. Specifically, the temperature of the rats in each group fluctuated over a range of 36~38℃, and there was no abnormal appearance in body temperature. Body temperature changes did not exceed 2℃, which varied in the normal fluctuation range.
Kim
The acute toxicity test results of this study on rBoIFN-α showed that even if the dose of bovine recombinant interferon alpha was as high as 50×1010 IU/kg b.w., it did not cause poisoning symptoms or death in the experimental rats, and after continuing to observe for another 14 days, it was found that the feeding and weight gain of the rats were normal, and the post-mortem observation showed that there were no abnormal changes in the heart, liver, spleen or other solid organs. In the maximum tolerance test, the rBoIFN-α was administered in doses of up to 50.000 g/kg and the rats were still alive and healthy at the end of the test. Their signs of breathing, visible mucosa, and fur were not abnormal, and post-medicine necropsy observation revealed that no substantial organs had visible characteristic pathological changes. The results suggested that rBoIFN-α had no toxic effect in rats, and could be expected to protect the health and safety of cattle.
The purpose of the sub-chronic toxicity test is to observe the toxic response of the animal caused by continuous repeated administration of the test drug, including the symptoms and severity of the occurrence and the primary organ evincing toxic effects and its recovery and development, and then to determine the non-toxic dose, according to the references for the safe clinical medication (1). The 30-day administration test in rats can be used as the main reference basis for evaluating the sub-chronic hazards of drugs or poisons. This experiment investigated the toxicity of rBoIFN-α by observing the clinical manifestations and histopathological changes, and determining haematological parameters, serum biochemical indexes, and organ indexes of rats after intravenous injection of drugs (10, 14). These latter included a relatively high gastrointestinal index for the middle- and high-dose groups. Considering the influence of the test animal indicators on the recipient weight, nutritional status, and other conditions, individual indicators might also have significant differences among groups. However, overall, after several-week-long administration of rBoIFN-α, there were no significant differences in rat body weight, blood haematology indicators, blood biochemical indicators, or organ indexes between experimental and control groups. Furthermore, no pathological changes related to drug effects were observed. These null findings implied that rBoIFN-α had no or extremely low toxicity.
It can be seen that use of rBoIFN-α over a sustained period had no significant effect on the haematological indexes of rats. Likewise it can be noted that administration of this recombinant interferon over the experimental duration did not influence serum biochemical indexes in rats. Several-week-long administration of rBoIFN-α induced only mild toxic effects or side effects on the main organs of rats and these effects did not usually injure the morphological structures of those organs.
Regarding the adverse drug reactions, some new findings have been made in experimental animals. For example, dark red discoloration and swelling of the liver were observed in two male rats. Congestion was observed in the ovary of a female rat in the control group and the 5×108 IU/kg group. However, these are individual phenomena and they ought not to be considered treatment-related changes. The incidence is very low and is independent of the dose level.
This study’s results showed that rBoIFN-α had no obvious toxic effect on test animals and is safe to use. During the test, the maximum dose was calculated according to kilogram body weight, which was equivalent to 1000 times the normal dose to cattle. Therefore, even as rBoIFN-α was administered in a 1000-fold larger dose than the clinical dose amount, it still had no noticeable acute toxic effect on rats. This result establishes a further experimental basis for the clinical use of rBoIFN-α.
The above results suggest that several-week-long administration of rBoIFN-α has rare adverse effects on the intake and body weight of rats. Besides, the several-week-long administration of rBoIFN-α seems not to cause substantial damage to the parenchymal organs of rats or induce significant abnormal changes. Therefore, this study suggests that rBoIFN-α seems to be safe for rats, and its use may foster the development of the cattle industry in China by protecting livestock health.
*Hai-Yang Yu and Dong-Mei Gao contributed equally to this study and should be considered co-first authors.