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Bull Vet Inst Pulawy 50, 537-542, 2006 EFFECT OF PARACETAMOL INJECTION IN OVO IN THE COURSE OF HATCHING AND THYROID HORMONE LEVELS IN CHICKEN EMBRYOS MARCIN W. LIS, ANDRZEJ SECHMAN1, JERZY W. NIEDZIÓŁKA AND JANUSZ RZĄSA1 Department of Animal Hygiene and Breeding Environment, 1 Department of Animal Physiology, University of Agriculture, al. Mickiewicza 24/28, 30-059 Kraków, Poland [email protected] Received for publication July 25, 2006. Abstract The aim of the study was to determine the effect of paracetamol (acetaminophen, P) on the course of embryogenesis and thyroid hormone (TH) levels in chicken embryos. Eggs from a broiler breeder flock were incubated under standard conditions. They were candled on the 5th d of incubation in order to discard unfertilized eggs and those with dead embryos. At the 6th d of incubation the eggs with living embryos were divided into 3 groups (n=120 in each); two of them were in ovo injected with P (4 mg/egg in 100 µl of aqua pro injectione) or with 100 µl of aqua pro injectione (control C1), while one group was non-injected (control C0). Thyroxine (T4), triiodothyronine (T3), and reverse-T3 (rT3) levels were measured radioimmunologically in samples of blood plasma collected from chick embryos on days: 11, 14, 16, 18, 20 (external piping), and on day 21 of incubation, i.e. just after hatching. The rest of the embryos were used for the investigation of hatchability parameters. In the P-treated group, a significant increase in the mortality of the embryos was found. It was mainly caused by the elevation of dead embryo number on the 6th and 7th d of incubation, i.e. just after P injection. In this group, much more damage to the liver and hyperplasia of the stomach were also observed. The time of hatching in P-treated chicks was significantly delayed. In comparison with both control groups, in P-treated embryos T4 was significantly higher on days 14, 16, and 18 of incubation, while it was considerably lower on day 20 and just after hatching. T3 concentrations in P-exposed embryos were significantly lower on day 11 and higher on day 14 and just after hatching. In P-injected embryos, rT3 levels were higher only on day 14. The results obtained indicate that paracetamol exerts unfavourable effects on chick embryogenesis; it decreases hatchability and delays hatching. This effect is probably associated with thyroid hormone alterations following P-treatment in late embryogenesis. Key words: chicken embryo, paracetamol, thyroid hormones, hatching. Paracetamol (acetaminophen; P) is a popular antipyretic and analgesic drug in human medicine. The antipyretic and analgesic effects of paracetamol are associated with a selective inhibition of the COX-2 cyclooxygenase and thereby synthesis of prostaglandin in the brain. P is absorbed completely from the digestive tract and the maximum concentration in blood is reached after 30-60 min following treatment. P is mainly metabolised in the hepatocytes and its half time in blood is about 2 h (16). As early as in 1966, Dikstein et al. (12) tried to use paracetamol in poultry breeding as the antihypertermic preparation and stimulator of the growth process in broiler chickens. P had been considered as a non-toxic medicine until 1966, when its hepatotoxicity was described (6, 10, and 31). The hepatotoxic effect is caused by one of P metabolites: N-acetyl-p-benzoquinone imine (NAPQI), which is able to evoke necrosis of hepatocytes (16). It has been established that paradoxically P is less harmful for children than adults are, because children have less intensive metabolism of hepatocytes (16). The hepatotoxic effect of P was also described in the case of cultured chick hepatocytes (19); however, the mechanism of this effect is not known. Laub et al. (17), using mouse embryos, showed that large doses of P did not affect the development of the preimplanted embryo. However, Lum and Wells (20) established that P administered to pregnant mice could be potentially teratogenic. Moreover, Stark et al. (28) observed that the addition of P to the medium of cultured rat embryos produced an increased incidence of abnormally open anterior neuropores. Abnormalities in cultured embryos, following P exposure, were also shown by Weeks et al. (34). However, they found that the N-acetylcysteine, a glutathione precursor, protects embryos from P toxicity. These results indicate that paracetamol affects mammalian embryonic development; however, in the literature there is no data concerning the effect of P on chicken embryogenesis. Therefore, the aim of the present study was to examine the consequence of paracetamol in ovo injection on chicken embryogenesis. Since thyroid hormones play a crucial role during this process and affect the time of 538 hatching, and the length of incubation (8, 14, 24-26), their concentrations following P injection in ovo were also investigated. Material and Methods Eggs (weighing on the average 60.2 ± 8.4 g) from the same broiler breeder flock of ISA 215 line were put to the incubator (Masalles 65 DIGIT) and incubated under standard conditions (1-18 d of incubation: t=37.8°C, RH=55%, 19-21 d of incubation; t=37.2°C, RH=70%). The eggs were candled on the 5th d of the incubation, and unfertilised eggs and those with dead embryos were discarded. On the 6th d of the incubation, the eggs with living embryos (n=360) were randomly divided into three equal groups. The experimental group was in ovo injected with 4 mg of paracetamol (POLFA-Kutno, Poland; Lot. No. 0255011) dissolved in 100 µl of aqua pro injectione (Polpharma, Poland). The administered dose of paracetamol was calculated based on the human acceptable daily dose (4 g P/60 kg b.w.). The eggs of the two control groups were injected with 100 µl of aqua pro injectione (control C1) or were not treated (control C0). The injections were given at the small end of the egg, deeply into albumin according to Borzemska et al. (4). During the incubation, the pathological analysis of dead embryos of each group was performed. The age, malformations, and malpositions of the embryo were estimated at the moment of death (5). Blood samples from each group were collected on days 11, 14, 16, 18, 20 (external piping), and on the 21st d of incubation, i.e. just after hatching. They were centrifuged 10 min (2 000 x g) and then plasma pools obtained from two to three embryos (days 11-20) and individual plasma samples from hatched chicks were kept in –20°C until hormone determination. Thyroid hormones (TH): thyroxine (T4), triiodothyronine (T3), and reverse-T3 (rT3) concentrations in plasma samples were measured radioimmunologically according to the method earlier described (1, 27). Standard solutions of T4, T3, and rT3 were prepared with chicken plasma free of endogenous thyroid hormones. Antibodies against T4 and T3 were obtained from Sigma (USA), while the antibody against rT3 from Professor Jerzy Kosowicz (University of Medical Sciences, Poznań, Poland). These antibodies have no or very weak cross reactivities with other iodothyronines. The radioactive iodothyronines were purchased from NEN (Belgium). The lowest limit of sensitivity for T4 and T3 was 1.25 and 0.08 ng/mL, respectively, while for rT3 – 5.4 pg/mL. The intra-assay coefficients of variation for T4, T3, and rT3 were 5.8%, 6.2%, and 4.0%, respectively. The hatchability and embryopathology were statistically analysed by z test, while the results of TH by two-way analysis of variance followed by Duncan’s multiple range test. The statistical analyses were performed using Sigma-Stat 2.03 (SPSS Science Software Ltd., USA). The figures were prepared with Grapher 4.0 (Golden Software Inc., USA). Because the radioimmunoassay revealed that there were no significant differences in plasma thyroid hormone levels between male and female embryos during incubation process, which is in agreement with previous findings (31), the data from both sexes were combined. The results were presented as means ± SEM. and considered significant at P<0.05 and highly significant at P<0.01. Results The results of the hatching and embryopathological analyses are presented in Tables 1 and 2, respectively; and changes in TH levels are shown in Fig. 1. A significantly higher mortality of embryos immediately after P injection (i.e. on days 6-7 of embryogenesis) occurred in paracetamol-treated group (P<0.01; Table 1). There were no significant differences in embryonic mortality between control and P-treated groups from the 8th d till the day of hatch; however, the total mortality of P-injected embryos was significantly (P<0.01) higher (27.1% in P-treated group vs. 20.0% and 21.2% in C1 and C0 groups, respectively; Table 1). The hatchability of chicken embryos treated with P was significantly (P<0.05) lower in comparison with the values of both control groups. In P-injected embryos the mean time of hatching was 507.2 ± 1.1 h and was significantly higher than in C0 group (P<0.05; Table 1). The pathological analysis of the dead embryos of each group, revealed that P injection in ovo significantly increased the liver damage in the form of lobe hypoplasia (or atrophia), hypertrophia of lateral lobes (lat. hypoplasia lobi hepatis et hypertrophia consecutiva loborum lateralium.), and subcapsular haemorrhages (lat. haermorrhagiae subcapsulares hepatis), and the gizzard damage (megagizzard) (P<0.05; Table 2). In the control groups (C1 and C0) plasma T4 concentrations increased gradually from 0.53±0.15 and 0.33±0.08 ng/mL on day 11 to the maximal values of 16.4±2.02 and 29.4±3.28 ng/mL on day of hatch, respectively (Fig. 1a). In embryos exposed to paracetamol, a similar increase in T4 concentration during embryogenesis was observed; however, on days 14, 16, and 18 the plasma levels of T4 for this group were by 165%, 51%, and 37%, respectively, higher in comparison with C1 (P<0.05). On day 20, a sharp decrease in T4 concentration in the P-treated group was found; the level of T4 on day 20 in P-injected embryos was 1.71 and 2.68 times lower in comparison with C1 and C0 groups, respectively (P<0.01). On the day of hatch, the level of T4 in the experimental chicks increased; however, it was still by 33% lower than that in non-injected chicks (C0 group, P<0.01, Fig. 1a). The plasma T3 concentrations gradually increased in both control groups (C1 and C0) from 0.32±0.07 and 0.14±0.08 ng/mL on day 11 to 3.68±0.60 and 3.14±0.17 ng/mL on day of hatching, respectively (Fig. 1b). In the embryos treated with paracetamol, the T3 concentrations on day 11 were 6.33 and 3.47 times lower than in C1 and C0 groups, respectively (P<0.01). 539 maximal levels of 167.1±18.2 and 225.3±15.1 pg/mL at the time of piping (day 20), respectively (Fig. 1c). Subsequently, the levels of rT3 sharply decreased toward hatching (54.7±7.2 and 32.2±6.1 pg/mL in C1 and C0 groups, respectively). In the P-treated group, the statistically (P<0.01) higher concentrations of rT3 in comparison with both control groups were found only on day 14 (increase by 233% and 131%, respectively). The significantly higher levels of rT3 in comparison with C0 (by 58%) and C1 (by 31%) groups were also noticed on days 18 and 20, respectively (P<0.05; Fig. 1c). On day 14, the concentrations of T3 increased and were by 88% and 79% higher than those in C1 and C0 groups, respectively (P<0.01). On day 18, the T3 level in the experimental group was 1.82 times lower than in the C0 group (P<0.05). On the other hand, on day of hatching, T3 concentrations in the P-treated chicks were by 37% and 62% higher in comparison with the values of C1 and C0 group, respectively (P<0.01, Fig.1b). The concentrations of rT3 on day 11 were below the sensitivity of the analytical method. In the control groups, (C1 and C0) plasma rT3 levels increased from 11.8±2.8, and 16.0±3.2 pg/mL on day 14, to the Table 1 Effect of paracetamol on the mortality and hatchability of chicken embryos Group Item: C1 (n*=80) C0 (n*=85) a Mortality - days 6-7 (%) Mortality – days 8-17 (%) Mortality – days 18-21 (%) Total mortality (%) Hatchability (%) Mean time of hatching ± SE (h) P (n*=70) b 4.7 4.7a 11.8a 21.2 a 78.8a 504.4±1.1a 15.7c 4.3a 7.1a 27.1b 72.9b 507.2±1.1b 6.3 3.8a 10.0a 20.0a 80.0a 505.1±1.0ab P- eggs injected with 4 mg of paracetamol on the 6th d of embryogenesis; C1 – eggs injected with aqua pro injectione; C0 – eggs non-treated; * - without eggs used for blood sampling a, b, c - values marked with different letters differ significantly (P<0.05) Table 2 Effect of paracetamol on frequency of malformations in chicken embryos mortalised between days 18 and 21 of incubation Item Embryos living at 18th d Type of detected malformations Malpositions Teratosis Liver damage: lobe hypoplasia (or atrophia) and hypertrophia of lateral lobes, subcapsular haemorrhages Megagizzard Incomplete retraction of the yolk sack C0 76 % 10.53a 3.95a Group C1 72 % 12.50a 2.78a P 56 % 8.93a 1.79a 1.32ab 0.00b 5.36a 1.32ab 9.21 0.00b 11.11 3.57a 7.14 a, b – values marked with different letters differ significantly (P<0.05). For further explanations, see Table 1. 540 (a) 40 C0 (non-treated) C1 (aqua pro injectione) Paracetamol (4 mg/egg) T4 [ng/ml] 30 b 20 ab 10 ab ab ab 0 E11 (b) E14 E16 E18 E20 H 6 ab T3 [ng/ml] 4 2 ab b ab 0 E11 (c) E14 E16 E18 E20 H 300 a rT3 [pg/ml] 200 b 100 ab 0 b.s. E11 E14 E16 E18 E20 H Fig. 1. Effect of paracetamol in ovo injected at the 6th d of incubation on plasma thyroxine (T4) (a), triiodothyronine (T3) (b) and reverse-triiodothyronine (rT3) (c) levels in embryonic chicks. Each value represents the mean ± SE from 5-7 determinations; a - P<0.05 in comparison with C1 (control injected with aqua pro injectione); b P<0.05 in comparison with C0 (control non-injected). 541 Discussion The present study showed that paracetamol in ovo injected at the first week of incubation affects the embryo development, decreases chick hatchability and delays the time of hatching. These unfavourable effects are accompanied by significant alterations in iodothyronines concentration in blood plasma of embryonic chicks. In the reported study, the embryotoxic effect of paracetamol was observed. This implication is confirmed by the significantly higher embryo mortality in the paracetamol treated group in comparison with both control groups at the first two days following injection. It can also be supported by the higher frequency of the embryo malformations caused especially by the elevation of the liver and stomach damage in the P-treated group. Although the death of the embryo after in ovo injection (as a manipulation) can often result from damage and disruption of the embryonic homeostasis, the applied substance is of more importance. Moreover, the embryos sensibility to the manipulation seems to be associated with the stage of its development (7). Many authors described the teratogenic effect (i.e., underdevelopment, heart damage, chorioallantoic haemorrhage, and malpositions) of growth stimulators, antibiotics, and other xenobiotics injected in ovo during the first week of chick embryogenesis (3, 4, 24, 32). Lin et al. (18) showed that injection of N-nitrozofenacitine (a derivative of paracetamol) in ovo on the 6th d of chicken embryogenesis evoked the feather and claw malformations, and caused brain damage. The toxic effect of paracetamol on the chicken hepatocytes in vitro has also been reported (19) but the mechanisms and circumstances, in which paracetamol damages hepatocytes, have been not described. However, it has been postulated that thyroid hormones can sensitize the liver cells to hepatic toxins. This hypothesis can be supported by the investigations of Hartleb et al. (13) who reported two cases of human liver damage mediated by paracetamol and accompanied by increased plasma level of thyroid hormones. In the performed experiment, a gradual increase in circulating iodothyronine concentrations observed during chicken embryogenesis under controlled conditions was consistent with data of the earlier studies (9, 22, 24, 26, 29, 30, 32). The lowest concentrations of T4 and T3 were found on day 11 of the chick embryogenesis, while the highest one was on the day of hatch when the chicken embryo had already been switched from chorioallantoic to lung respiration (9, 11). The plasma T4 concentrations were low until day 16 of embryogenesis, and then it rose exponentially toward the day of hatch. The most outstanding finding of this study was the effect of paracetamol on iodothyronine concentration in blood plasma. Paracetamol increased T4 concentration in the middle phase of embryogenesis (i.e. between the 14th and 18th d); however, during the hatching period, T4 concentration was significantly lower in paracetamol-treated chicken embryos. These changes were accompanied by a significant delay of the hatching process in paracetamol treated group. The correlation between the thyroid hormones concentration in blood plasma and a rate of hatching was also observed in chicken embryos exposed to dioxin-like polychlorinated biphenyls (24) and electromagnetic field (26). It seems likely that the observed changes in T4 concentration in blood of chicken embryos following paracetamol treatment are probably associated with a negative effect of this preparation on the hypothalamopituitary-thyroid axis. It cannot be excluded that the decrease in T4 during the last phase of embryogenesis, is connected with a competitive effect of paracetamol compound with T4 binding to blood transport proteins involved in the distribution of thyroid hormones in the organism (15, 23). In the case of T3, a sharp increase in concentration of this hormone in paracetamol-injected embryos was found at the day of hatch. It may resemble both the function of the thyroid gland, which secretes T3 (2, 21) and the activity of the hepatic and renal iodothyronine deiodinases: type I (D1), which catalyses both outer (ORD) and inner (IRD) ring deiodination, and type III (D3) that is responsible for the IRD of T4 and T3 into 3.3’,5’-triiodothyronine (rT3) and 3,3’diiodothyronine (3.3’-T2), respectively (8, 9, 22, 32). The gradual increase in rT3 concentration before hatching is probably a result of the high activity of deiodinase D3 in the liver (8, 9). It cannot be excluded that alterations in iodothyronines concentration in blood plasma of paracetamol-treated embryos are also associated with the hepatotoxic effect of this preparation. In conclusion, the results obtained indicate that paracetamol exerts unfavourable effects on chick embryogenesis: (i) increases embryo mortality; (ii) decreases hatchability, in addition, (iii) delays the time of hatching. 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