Introduction
Acetaminophen, also known as paracetamol, is extensively used as an analgesic and antipyretic agent in human1 and veterinary medicine. Acetaminophen is used for the treatment of painful disease states associated with fever in swine2 and, occasionally, in the control of postoperative pain in dogs in association with codeine.3–5 In some countries, it is administered to cattle for fermentation disorders and acetonemia and in poultry for the treatment of painful diseases and pyrexia.2,6,7 Only a few reports on acetaminophen (APAP) pharmacokinetics in poultry are present in the literature. Its pharmacokinetics were reported following a single oral dose of 10 mg/kg to chickens and turkeys and after an IP injection of 100 mg/kg in chicks.8,9 No residue depletion studies for poultry have been described.
Although globally geese are of relatively minor importance, goose breeding is increasing in several Asian and European countries including China, Hungary, and Poland.10 Little information on potential therapeutic treatments for the management of diseases associated with fever or pain is available for this species.6,11–13 APAP demonstrated good antinociceptive properties in pigeons.6 Additionally, it seems to be safer than diclofenac or other NSAIDs in poultry since no nephrotoxic signs were observed after a 10-mg/kg IM injection daily for 7 days.14 To the best of the authors’ knowledge, no previous studies have been performed to investigate the pharmacokinetic features of APAP in geese. Since data extrapolation from other species and breeds may be inaccurate or dangerous due to species- and breed-specific differences,15–18 the aims of the study were to evaluate the pharmacokinetics of APAP after IV and PO administration of a single dose (10 mg/kg) in geese and to quantify APAP and its main metabolites (acetaminophen glucuronide [PG] and acetaminophen sulfate [PS]) in goose tissues. Moreover, a histopathologic evaluation of goose stomach, duodenum, liver, and kidney tissues was carried out after a single PO administration of APAP (10 mg/kg) to observe potential signs of toxicity.
Materials and Methods
Chemicals and reagents
APAP, PS potassium, PG sodium salt, and 4-acetamidophenyl β-D-glucuronide-d3 sodium salt (internal standard) were used in this study (Sigma-Aldrich). Acetonitrile, methanol, and SiOH 500-mg solid-phase extraction cartridges were also used (Avantor Performance Materials). Formic acid (99%) was acquired from VWR Chemicals, and distilled water (UltraPure; Invitrogen) was obtained using a purification system (Milli-Q; Millipore).
Animal treatment
Twenty-four adult male geese (Anser anser domesticus) were randomly selected from a larger flock and randomly divided in 3 groups by use of a software program19; each group was composed of 8 animals. Body weight and age of the geese ranged from 4.6 to 5.5 kg (average age, 5.1 kg) and 3 to 4 years (average age, 3.6 years), respectively. The animal experiment was approved by the Institutional Animal Care and Use Committee of the University of Lublin (Poland) and carried out in accordance with European law (2010/63/UE). The geese were judged to be in good health based on physical examination before the beginning of the study and were monitored during the experimental trial through daily observation of behavior and appetite. Birds were acclimatized for 2 weeks into a 60-m2 enclosed area with an indoor shelter of 8 m2 before the beginning of the study. Geese were fed with a drug-free pelleted diet twice a day and water ad libitum. Animals could graze freely during the day and were identified using an identity code ring applied on the right leg.
Group I was administered APAP (paracetamol, B. Braun; solution for infusion at 10 mg/mL) at 10 mg/kg in the left brachial wing vein using a sterile 26-gauge, 1.75-cm needle.
Group II was treated PO with APAP (500-mg paracetamol tablets; Accord) at the same dosage by crop gavage 3 hours after being fed. The marketed drug was ground, homogenized, partitioned, and dosed according to the body weight of each bird. The correct weight of the powder was dissolved in 2 mL of water for easy administration using a rounded tip metal catheter. Approximately 1 mL of blood was collected in lithium heparin tubes at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 24 hours and 0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 24 hours after IV and PO administration, respectively, using a 24-gauge catheter inserted, after the manual removal of a few feathers, in the right brachial wing vein and fixed with adhesive bandage tape. The catheter was flushed with 1 mL of saline (0.9% NaCl) solution with the addition of heparin (10 IU/mL) at each collection time point, and the first 0.2 mL of blood was discarded before each blood sample withdrawal. Tubes were centrifuged at 1,500 X g, and the plasma was harvested and stored at −20 °C until further analysis.
Group III was administered APAP (10 mg/kg) PO as previously described for group II. Two animals were humanely euthanatized by stunning and exsanguinated at each selected time points (1, 4, 10, and 24 hours) for tissue collection. A portion of stomach, duodenum, liver, and kidney was removed and prepared for the histopathological evaluation. A portion of muscle, heart, lung, liver, and kidney was collected at the same time points for the quantification of APAP and its metabolites.
Drug and metabolites extraction and quantification
APAP and its metabolites were quantified using an ultra–high-performance liquid chromatography–tandem mass spectrometry method previously developed and validated in tissues by Pietruk et al.20
Briefly, 2 g of heart, muscle, liver, lung, or kidney sample was spiked with 10 µL of internal standard working solution (5 µg/mL). Then, 4 mL of acetonitrile and 4 mL of 0.1% formic acid in methanol were added and vortexed. The sample was centrifuged for 10 minutes at 20 °C, and 6 mL of the supernatant was passed through a C18 solid-phase extraction cartridge that was preconditioned with 2 mL of methanol. The filtrated extract was dried using a nitrogen stream at 45 °C. The residue was dissolved in 600 µL of 0.1% formic acid and filtered into vials. Ten microliters of this latter solution was injected into the chromatography system.
Two hundred microliters of plasma was placed into the microcentrifuge tube, fortified with 40 µL of internal standard at a concentration of 1 µg/mL. Then, 460 µL of methanol was added, mixed for 30 seconds, and centrifuged for 5 minutes. Six hundred microliters of supernatant was transferred into a glass tube and evaporated to dryness under a stream of nitrogen at 45 °C. The dry residue was then reconstituted with 300 µL of 0.1% formic acid and transferred into liquid chromatography vials before injection into the chromatography system.
The liquid chromatography tandem–mass spectrometry analysis was performed with an ultra–high performance liquid chromatography system (Nexera X2; Shimadzu) connected to a triple quadrupole mass spectrometer (SCIEX 4500; Sciex). Separation of the analytes was performed using a 50 X 2.1-mm, 1.8-µm column (Zorbax RRHD; Agilent Technologies Inc) maintained at 45 °C coupled with a guard column. The mobile phase composition was 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). Gradient elution was performed with the following program: 0 to 5 minutes 95% A, 5 to 6.3 minutes 15% A, and finally from 6.31 to 8 minutes back to 95% A at a flow rate of 0.6 mL/min. Detection was conducted in positive and negative electrospray ionization mode. Two transitions were monitored for paracetamol and metabolites and 1 transition for the internal standard (Analyst software version 1.6.2; Sciex).
Method validation
The method for the determination of APAP and its metabolites in goose plasma was validated based on linearity, selectivity, precision, recovery, limits of detection, and quantification. Two different calibration curves (10 to 500 ng/mL and 1,000 to 10,000 ng/mL) were prepared using drug-free goose plasma samples spiked at 5 concentration levels. The specificity was evaluated by analyzing different blank goose plasma samples (n = 6) and checked for potential interferences with endogenous substances. Precision, repeatability, and within laboratory reproducibility were calculated by the repeated analysis of drug-free plasma samples (n = 6) fortified with APAP and its metabolites at 3 concentration levels (50, 100, and 250 ng/mL). The coefficients of variation were calculated. For repeatability, samples were analyzed on the same day by the same operator and with the same instrument. For within laboratory reproducibility, another 2 sets of fortified samples at the same concentration levels as for the repeatability were analyzed on 2 different days with different operators and the same instrument. The average recovery was investigated by comparing the mean measured concentration with the fortified concentration of the samples. The limits of detection and quantifications were calculated as a signal-to-noise ratio of 3 and 10, respectively, of each analyte in fortified (lowest detectable concentration level) samples. Validation results for tissues have been reported previously by Pietruk et al.20
Histopathological evaluation
Immediately after euthanasia of the animals, a portion of proventriculus (glandular stomach), gizzard (muscular stomach), duodenum, liver and kidney were collected. The material was rinsed with saline solution, fixed in neutral-buffered 10% formalin, and embedded in paraffin blocks using a routine histological technique. Ten-micrometer-thick sections of the selected organs were obtained using a microtome and placed on slides (SuperFrost Plus; Fisher Scientific). Then, all sections were stained with Mayer H&E. The stained slices were observed, analyzed with a light microscope (Olympus BX51; Olympus), and photographed (Digital Olympus Color View III camera; Olympus).
Pharmacokinetic analysis
The pharmacokinetic analysis was performed with pharmacokinetic software (ThothPro version 4.3; ThothPro). A noncompartmental approach was used for data evaluation obtained after IV and PO administration of APAP. Maximum concentration (Cmax) and time at maximum plasma concentration (Tmax) of APAP, PG, and PS were determined from the raw data. The elimination half-life was calculated by linear regression on the log-transformed concentration data in the terminal phase. The area under the curve (AUC) of APAP was calculated by linear log trapezoidal and the linear-up log-down rule to the final concentration–time point for the IV and PO group, respectively. From these values, APAP volume of distribution (Vss = dose X area under the first moment curve/AUC2) and systemic clearance (clearance = dose/AUC) were determined. The mean residence time = area under the first moment curve/AUC. The absolute oral bioavailability (F) was calculated as:
Individual values between the area under the concentration–time curve from 0 to infinity and area under the concentration-time curve from 0 to the last detectable time point were lower than 20% of the area under the plasma concentration–time curve from the last measured time extrapolated to infinity and expressed as a percentage of the total area under the plasma concentration–time curve (< 20%), and R2 was > 0.85. The extraction ratio for APAP after IV administration was calculated for each bird as clearance divided by cardiac output,21,22 where cardiac output (mL/min) was calculated as body weight (kg) to the power of 0.69 multiplied by 290.7.21
A naïve pooled-data approach using a noncompartmental analysis23 was used to calculate the pharmacokinetic parameters for APAP and its metabolites in all the selected tissues.
The drug tissue accumulation was determined considering the ratios between the AUC value found in each tissue and in plasma after PO administration.
Statistical analysis
All pharmacokinetic values are presented as geometric mean and range,24 while Tmax as a categorical variable, was expressed as median and range. Statistical analysis was performed using a statistical software (GraphPad Prism 5.0 version; GraphPad Software Inc). Mean values were compared between the 2 routes of administration using Wilcoxon rank-sum test.
Results
Validation results
The developed method was successfully validated in goose plasma, and the matrix-matched curves exhibited good linearity for all the analytes through the coefficient of correlation r2 > 0.996. The method specificity showed that no potential interfering compounds were detected at the retention time of the APAP and metabolites. The average recovery and the coefficients of variation for reproducibility and within-laboratory reproducibility are reported (Table 1). The limit of detection was 5 ng/mL, and the limit of quantification was 10 ng/mL for all analytes.
Validation parameters of the analytical method used for the quantification of acetaminophen (APAP), acetaminophen glucuronide (PG) and acetaminophen sulfate (PS) in goose plasma.
| Analyte | Repeatability † (CV%) | Within-lab reproducibility† (CV%) | Limit of detection (ng/mL) | Limit of quantification (ng/mL) | Recovery† (%) |
|---|---|---|---|---|---|
| APAP | 7.7 ± 2.6 | 11.3 ± 1.8 | 5.0 | 10.0 | 87.4 ± 4.6 |
| PS | 9.2 ± 2.3 | 13.2 ± 2.8 | 5.0 | 10.0 | 111.2 ± 3.7 |
| PG | 11.7 ± 2.7 | 14.8 ± 3.1 | 5.0 | 10.0 | 92.8 ± 5.1 |
†Mean ± SD (n = 6) for each validation level.
CV = Coefficients of variation.
Pharmacokinetics
Mean plasma concentrations of APAP and its main metabolites versus time after IV and PO administration of APAP at 10 mg/kg were determined (Figure 1). APAP plasma concentrations were lower compared to those of the metabolites at almost all of the selected time points after both treatments. It is noteworthy to report that a secondary-peak was observed in plasma APAP concentration 4 hours after IV administration in all the animals.
Mean acetaminophen (APAP), acetaminophen glucuronide (PG), and acetaminophen sulfate (PS) plasma concentration versus time curve after a single 10 mg/kg IV (A) and PO (B) administration of APAP in geese (n = 8).
Citation: Journal of the American Veterinary Medical Association 260, 12; 10.2460/javma.21.05.0250
The pharmacokinetic parameters obtained from the IV and PO data analysis are listed (Table 2). After IV treatment, the AUCAPAP value was significantly (P < 0.001) higher than that found after PO administration, resulting in an oral bioavailability of 46%. In contrast, PO AUCPS, and AUCPG were statistically higher (double) than those found after IV administration (P < 0.001).
Main pharmacokinetic estimates (geometric mean) of APAP, PG, and PS in geese after a single IV (n = 8) and PO (n = 8) APAP administration (10 mg/kg).
| Parameter | Unit | APAP | |||||
|---|---|---|---|---|---|---|---|
| IV | PO | ||||||
| Geometric mean | Minimum | Maximum | Geometric mean | Minimum | Maximum | ||
| AUC(0–t) | μg • h/mL | 24.91 | 20.61 | 33.92 | 10.52*** | 8.37 | 12.89 |
| AUC(0–inf) | μg • h/mL | 24.95 | 20.65 | 33.95 | 11.55*** | 9.23 | 13.36 |
| MRT(0–t) | h | 3.08 | 2.65 | 3.49 | 2.31*** | 1.71 | 2.78 |
| MRT(0–inf) | h | 3.11 | 2.70 | 3.51 | 4.04 | 2.43 | 15.86 |
| kel | 1/h | 0.15 | 0.13 | 0.19 | 0.14 | 0.02 | 0.35 |
| t1/2kel | h | 4.66 | 3.70 | 5.33 | 5.01 | 1.97 | 29.60 |
| Cmax | μg/mL | — | — | — | 5.31 | 4.37 | 6.50 |
| Tmax† | h | — | — | — | 0.25 | 0.25 | 0.50 |
| Cl | mL/g h | 0.40 | 0.30 | 0.49 | — | — | — |
| Vss | mL/g | 1.23 | 1.03 | 1.36 | — | — | — |
| F | % | — | — | — | 46.29 | 34.49 | 59.56 |
| Parameter | Unit | PG | |||||
|---|---|---|---|---|---|---|---|
| IV | PO | ||||||
| Geometric mean | Minimum | Maximum | Geometric mean | Minimum | Maximum | ||
| AUC(0–t) | μg • h/mL | 48.09 | 37.17 | 64.45 | 89.04*** | 61.73 | 108.50 |
| AUC(0–inf) | μg • h/mL | 48.14 | 37.17 | 64.67 | 93.06*** | 71.14 | 108.92 |
| MRT(0–t) | h | 3.51 | 2.97 | 4.27 | 4.68** | 3.43 | 5.50 |
| MRT(0–inf) | h | 3.54 | 2.97 | 4.32 | 5.30*** | 4.95 | 5.69 |
| kel | 1/h | 0.30 | 0.23 | 0.36 | 0.20*** | 0.18 | 0.24 |
| t1/2kel | h | 2.34 | 1.94 | 3.06 | 3.41*** | 2.87 | 3.90 |
| Cmax | μg/mL | 12.96 | 10.90 | 18.50 | 16.55 | 13.40 | 21.40 |
| Tmax† | h | 1.00 | 0.50 | 1.50 | 0.75 | 0.75 | 1.50 |
| Parameter | Unit | PS | |||||
|---|---|---|---|---|---|---|---|
| IV | PO | ||||||
| Geometric mean | Minimum | Maximum | Geometric mean | Minimum | Maximum | ||
| AUC(0–t) | μg • h/mL | 31.46 | 22.22 | 60.72 | 53.02** | 38.42 | 79.18 |
| AUC(0–inf) | μg • h/mL | 31.56 | 22.30 | 60.74 | 57.44*** | 48.31 | 80.11 |
| MRT(0–t) | h | 2.56 | 2.09 | 3.19 | 3.96*** | 2.66 | 4.85 |
| MRT(0–inf) | h | 2.64 | 2.18 | 3.65 | 5.32*** | 4.25 | 11.65 |
| kel | 1/h | 0.23 | 0.14 | 0.33 | 0.17 | 0.05 | 0.27 |
| t1/2kel | h | 3.02 | 2.10 | 4.94 | 4.13 | 2.59 | 14.07 |
| Cmax | μg/mL | 13.66 | 10.80 | 18.30 | 13.57* | 11.13 | 20.10 |
| Tmax† | h | 0.38 | 0.25 | 1.00 | 0.50 | 0.25 | 1.50 |
Significantly different between the treatments:
P < 0.05;
P < 0.01;
P < 0.001. †Median value.
— = Not applicable. AUC(0–inf) = Area under the concentration–time curve from 0 to infinity. AUC(0–t) = Area under the concentration–time curve from 0 to the last detectable time point. Cmax = Maximum concentration. kel = Elimination rate constant. MRT(0–inf) = Mean residence time from 0 to infinity. MRT(0–t) = Mean residence time from 0 to the last detectable time point. t1/2kel = Elimination half-life. Tmax = Time at maximum plasma concentration. Vss = Volume of distribution at the steady state. Cl = Clearance.
See Table 1 for remainder of key.
Tissue residue analysis
The residues of APAP and its main metabolites in goose muscle, heart, liver, kidney, and lung tissues are shown (Figure 2) after a single PO administration (10 mg/kg). APAP residues were higher in the liver and lung compared to the other tissues with higher AUC and Cmax values. In the liver and lung, the tissue accumulation ratio was the highest (Table 3). In the liver, PS concentrations were very close to the limit of quantification, while it was the highest in the lung. The AUCPG and AUCPS values were similar in the lung, while in the liver, AUCPG was about 30-fold higher than AUCPS. The kidney showed low drug and metabolite levels, but the PG residue was much higher compared to APAP and PS levels. The APAP and PS levels were quantifiable only for the first 2 collection time points (1 and 4 hours), and their pharmacokinetic parameters could not be calculated. APAP concentrations in the heart and muscle were similar.
Mean APAP, PG, and PS tissue concentration versus time curve after a single PO administration (10 mg/kg) of APAP in goose muscle (A), heart (B), liver (C), kidney (D), and lung (E) (n = 2/time point).
Citation: Journal of the American Veterinary Medical Association 260, 12; 10.2460/javma.21.05.0250
Pharmacokinetic parameters (geometric mean) for APAP, PG, and PS in different tissues following PO administration of APAP to geese (n = 2/time point) at a dose of 10 mg/kg.
| Parameter | Unit | Matrix | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Muscle | Heart | Liver | Kidney | Lung | ||||||||||||
| APAP | PG | PS | APAP | PG | PS | APAP | PG | PS | APAP | PG | PS | APAP | PG | PS | ||
| AUC(0–t) | μg • h/mL | 11.30 | 5.65 | 10.30 | 9.21 | 14.19 | 9.10 | 32.38 | 19.76 | 0.67 | — | 11.84 | — | 24.64 | 41.33 | 42.50 |
| Cmax | μg/mL | 2.63 | 0.86 | 2.63 | 2.44 | 2.39 | 1.35 | 6.40 | 3.70 | 0.09 | — | 1.73 | — | 5.49 | 6.28 | 6.44 |
| Tmax† | h | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 2.50 | — | 2.50 | — | 1.00 | 1.00 | 1.00 |
| Accumulation index | 1.06 | 0.06 | 0.19 | 0.86 | 0.16 | 0.17 | 3.04 | 0.22 | 0.01 | — | 0.13 | — | 2.31 | 0.46 | 0.80 | |
Histopathological results
No histological alterations in the morphology of stomachs, duodenal wall, and kidneys were observed in any of the examined birds (data not shown). The microscopic analysis showed no differences in the structure of the goose liver from the experimental groups compared to the control animals in the first hours after administration. A normal and typical structure of the liver was found in all the geese at 1, 4, and 10 hours after drug administration. However, the goose liver samples collected at 24 hours after drug administration showed dilated capillaries and lipid accumulation in hepatocytes (Figure 3).
Fragment of the hepatic lobule from the control group (A) and treated group 24 hours after administration of APAP (B; 10 mg/kg). Notice the central vein (asterisk), sinusoids (arrowhead), and hepatocytes (arrow). Magnification = 200X. H&E stain; bar = 50 µm.
Citation: Journal of the American Veterinary Medical Association 260, 12; 10.2460/javma.21.05.0250
Discussion
This study provides information regarding APAP pharmacokinetics and its residues in selected tissues. A histopathological analysis was performed to assess for potential signs of toxicity, as liver necrosis and tubular nephrotic changes were previously observed in chickens after a single PO dose of 20 or 40 mg/kg.25
APAP pharmacokinetics have been previously investigated in avian species such as chickens and turkeys.8 PG and PS plasma levels were evaluated in turkeys only and were always higher than APAP concentrations. A similar situation was observed in the present research. APAP was eliminated (elimination half-life = 4.66 hours) at a slower rate than in chickens (0.61 hours) and turkeys (0.67 hours).8 Vss (1.23 mL/g) in geese was in line with those found in other avian and in some mammalian species.7,8,26,27 APAP is a small lipophilic molecule with a low molecular weight, and it is found in a unionized form at all physiological pH values.27,28 These features, including the low plasma protein binding (15%, swine; 21%, humans),29 may contribute to its wide distribution. Clearance in geese (0.40 mL/g • h) was slower compared to those reported in chickens (1.89 mL/g • h) and turkeys (1.98 mL/g • h), and the E value was found to be low (0.05) as extrapolated from other studies in poultry (chickens, E = 0.1; turkeys, E = 0.08).8
APAP Cmax (5.31 µg/mL) and Tmax (0.25 hours) resulted in an intermediate value between those reported in chickens (8.61 µg • h/mL; 0.17 hours) and turkeys (1.07 µg • h/mL; 0.77 hours) administered the same dose.8 The difference in gastric emptying and feed status between the studies may have influenced these parameters.30 Moreover the different formulation administered, different excipients and carrier, may be other potential factors of this variation.
AUC of APAP was statistically different between the PO and IV treatments. Even if AUC (24.95 µg • h/mL, IV; 11.55 µg • h/mL, PO) was higher in geese compared with those found in chickens (16.18 µg • h/mL, IV; 6.70 µg • h/mL, PO) and turkeys (5.37 µg • h/mL, IV; 2.20 µg • h/mL, PO,), the oral bioavailability was moderate (46%) and in line with those from other avian species (chickens, 42%; turkeys; 39%).8 This suggests that although species specific differences may occur in drug exposure, the bioavailability of the drug is similar. Species-specific differences in the absorption process and the different formulation used may have contributed to the discrepancy in AUC values between the above-mentioned avian species. For instance, in the study by Neirinckx et al,8 the IV formulation was also used for the oral administration.
Some differences have been reported in the AUC and/or F value of APAP between avian and mammalian species (turkeys, chickens, geese vs pigs, and horses).8 These may be related to species-specific differences in the extent of the first-pass hepatic extraction or in the absorption process.8,31 Indeed, in most animal species, humans included, APAP is rapidly and predominantly absorbed from the gastrointestinal tract, but it is not completely available to the systemic circulation due to the first-pass metabolism.8,31–33 Other metabolic processes have been documented to occur in the intestine during absorption.34,35
APAP exhibited a secondary peak in plasma profile 4 hours after IV administration in all the geese. Surprisingly, this peak did not occur after PO treatment nor in metabolite concentration profiles. It might be attributed to the phenomenon of enterohepatic recycling.30,36,37 Although this is the first time that enterohepatic circulation is speculated to be relevant in APAP metabolism in geese, other NSAIDs such as rofecoxib, carprofen, and diclofenac undergo enterohepatic recirculation in veterinary species.38–42 In addition, a secondary peak concentration after IV administration of rofecoxib and diclofenac has been reported in rats.38,42
After PO administration, AUCPG/AUCAPAP (1.93) and AUCPS/AUCAPAP (8.06) ratios were higher compared to those found after IV administration (1.27 and 4.97, respectively). This may be related to some enzymes being present in the intestine, which may have a different influence on the oral formulation administered.34,35 These IV ratios (metabolite/APAP) are not in line with the results reported in goats and camels.27 It should be noted that this comparison is being made for significantly different species (avian vs mammals) and for data obtained with different analytical methods.43
Tissue residue analysis
On tissue residue analysis, higher APAP concentrations were found in the liver and lung, compared to muscle, heart, and kidney. Tissue accumulation occurred in liver and lung, with an accumulation index of 3.04 and 2.31, respectively. Regarding liver, this might be related to the fact that APAP is metabolized predominantly in the liver and/or to the possible covalent binding of APAP to hepatocytes.1
As far as the lung is concerned, APAP seemed to penetrate quite well in goose lungs. This behavior might be related to the physiological and anatomic species characteristics and to the physicochemical properties of the drug: it may lead to a high affinity to the alveolar epithelial lining fluid and/or bronchoalveolar lavage cells as it happened for other drugs such as tulathromycin.44 Although few studies are available in the literature on the effects of APAP on human lungs, it has been speculated that APAP may be present at this site.45–47 However, no information is available on lung residues in avian species and the explanation for its high accumulation index is uncertain and needs further investigations.
Higher PS concentrations were found in the kidney compared to APAP. This is in line with early studies of animal species and humans in which APAP is renally eliminated (approx 90% in humans) mostly as metabolites, while only 2% to 5% of the unchanged drug is excreted in the urine.8,27,31,32
Histopathological results
No adverse effects were observed during or after the experimental trial in any of the animals. No signs of toxicity were found in the kidney which is the main site of toxicity for NSAIDs.48–50 This is in line with the results of Jayakumar et al,14 in which no histopathological changes were found in chicken kidneys after multiple 10-mg/kg administrations. Severe hepatotoxicity signs with an increase in liver AST, ALT, and ALP were observed in chickens after APAP was administered at 650 mg/animal, while a dose of 2 g/kg was found to be lethal.51,52 In the present study, minor liver alterations were observed in both geese liver samples collected at 24 hours after administration, suggesting fatty degeneration of hepatocytes. Oxidative stress caused by the toxic metabolite of APAP (N-acetyl-p-benzoquinone) might be responsible for these effects. Unfortunately, in the present study it was not possible to obtain data related to the toxic metabolite N-acetyl-p-benzoquinone, limiting the reliability of this latter speculation. However, consistent with the present results, signs of liver congestion were previously observed in poultry after a single dose of APAP (10 mg/kg), while severe alterations such as progressive granular degenerative changes and diffuse or focal necrosis were reported in poultry treated with higher dosages (20 and 40 mg/kg).25 The toxicity evaluations performed in the present research cannot lead to a rigorous conclusion since multiple dose and efficacy studies are required to better assess the severity of hepatotoxicity at the therapeutic dose (unknown yet) in geese. Thus, it can also be concluded that doses higher than 10 mg/kg should be avoided since mild or severe hepatotoxic effects may occur.
Acknowledgments
Supported by the University of Pisa.
The authors thank ThothPro for supplying the software used for the pharmacokinetic analysis and Professor Helen Owen of University of Queensland for the scientific and English editing of the manuscript.
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.
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