Effects of Phenoxyacetic Acid Herbicides on Chicken Embryo Liver Drug Metabolizing Enzymes

Abstract: Chick embryos were treated on day 0 of incubation with two phenoxy herbicides, 2-methyl-4-chlorophenoxyacetic acid (MCPA) (0.4, 2 mg/egg) and 2,4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 4 mg/egg). Both herbicides seemed to exert toxic effects mainly on the liver of 19-day-old embryos. Specific histological analysis indicated biliary stasis. Ethoxycoumarin O-deethylase was depressed by MCPA but raised by 2, 4-D. Other hepatic monooxygenase activities were unaffected by the herbicides and no significant changes were found in cytochromes. The higher dose of MCPA increased NADPH-cytocrome P₄₅₀ reductase. 2,4-D treatment increased by activity of glutathione-S-transferases in the hepatic post-microsomal fraction while MCPA increased them at the lower dose and significantly reduced them at the higher. The phenoxyacetic herbicides appear thus to have some effects on hepatic drug metabolizing enzymes of the chick embryo which cannot be easily interpreted. Biliary retention, produced in particular by MCPA, could be partly responsible for these effects.     

Effects of phenoxyacetic acid herbicides on chicken embryo liver drug metabolizing enzymes.

Chick embryos were treated on day 0 of incubation with two phenoxy herbicides, 2-methyl-4-chlorophenoxyacetic acid (MCPA) (0.4, 2 mg/egg) and 2,4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 4 mg/egg). Both herbicides seemed to exert toxic effects mainly on the liver of 19-day-old embryos. Specific histological analysis indicated biliary stasis. Ethoxycoumarin O-deethylase was depressed by MCPA but raised by 2, 4-D. Other hepatic monooxygenase activities were unaffected by the herbicides and no significant changes were found in cytochromes. The higher dose of MCPA increased NADPH-cytochrome P450 reductase. 2,4-D treatment increased by activity of glutathione-S-transferases in the hepatic post-microsomal fraction while MCPA increased them at the lower dose and significantly reduced them at the higher. The phenoxyacetic herbicides appear thus to have some effects on hepatic drug metabolizing enzymes of the chick embryo which cannot be easily interpreted. Biliary retention, produced in particular by MCPA, could be partly responsible for these effects.     

Influence of hypothyroidism induced by thiamazole on the toxic interaction between propranolol and disopyramide in chick embryos

The effect of the hypothyroidism induced by thiamazole on toxic interactions between propranolol and disopyramide were studied in chick embryos. Fertilized eggs of White Leghorns were incubated and investigated. 1.2 mg/0.2 ml/egg of thiamazole was injected into the albumen of fertilized eggs on the 9th day of incubation. The control group was given 0.2 ml/egg of physiological saline in the same manner. Propranolol at 0.1 mg/egg and disopyramide at 0.3 mg/egg were injected into the air sac of fertilized eggs on the 16th day of incubation. Electrocardiograms were recorded 0 to 60 min after the injection. After the injection of propranolol and disopyramide into the thiamazole treated eggs, the heart rate was significantly decreased compared with the thiamazole untreated eggs. These findings indicate that hypothyroidism induced by thiamazole has a marked influence on the toxic interaction between propranolol and disopyramide in chick embryos.     

Influence of hypothyroidism induced by thiamazole on the toxicity of amitriptyline in chick embryos

The effect of hypothyroidism induced by thiamazole on the toxicity of amitriptyline was studied in chick embryos. Fertilized eggs of White Leghorns were incubated and investigated. 1.2 mg/0.2 ml/egg of thiamazole was injected into the albumen of fertilized eggs on the 9th day of incubation. The control group was given 0.2 ml/egg of physiological saline in the same manner. Amitriptyline at 1 mg/egg was injected into the air sac of fertilized eggs on the 16th day of incubation. Electrocardiograms were recorded 0 to 60 min after the injection. After the injection of amitriptyline into the thiamazole-treated eggs, the heart rate was significantly decreased compared with the untreated eggs. These findings indicate that hypothyroidism induced by thiamazole has a marked influence on the toxicity of amitriptyline in chick embryos.     

Developmental stage dependent toxicity of aminoguanidine on chick embryos

Aminoguanidine (AG) sulfate was injected into the thin albumen of fertile eggs on the different days of incubation. The lethality of AG sulfate on the developing chick embryos became lower when the treatment was done at later developmental stage. When AG was injected on the 5th day of incubation, the frequency of retardation of development of the body and liver, aplasia of gallbladder and enlarged spleen was highest without high mortality compared with the other experiments. Embryos with liver damage could not hatch. 14C-AG hydrochloride injected on the 5th day of incubation easily distributed into the embryo through the yolk from the thin albumen of the injection site, but when injected on the 9th day, the agent transferred very little into the yolk and embryo. The change of transport in the eggs with development may elucidate the difference of lethality of AG. Additionally, it was confirmed using thin layer chromatography that a large part of the radioactivity in the embryo might be an unknown metabolite of AG and the substance was accumulated with extremely high concentration in the liver at 24-48 hr after the injection of AG on the 5th day of incubation. This is probably the main cause of the peculier abnormalities in the liver of chick embryos.     

Effects of in ovo injection of carbamates on chick embryo hatchability, esterase enzyme activity and locomotion of chicks

Carbaryl and aldicarb, two carbamate pesticides used extensively throughout the United States, are known to act as acetylcholinesterase inhibitors. We have demonstrated previously that exposure to carbaryl and aldicarb in young chicks caused persistent locomotion alterations with no correlation to esterase inhibition. In this study, we investigated the effects of these carbamates when injected in ovo to chick embryos, at two time periods (days 5 and 15) during incubation. Carbaryl dosed at 45 mg kg-1 egg weight was extremely toxic to the embryos on day 5 of incubation. Hatchability was reduced to 0% as compared to 80% when carbaryl was injected on day 15 of incubation. Aldicarb at 1.5 mg kg-1 egg weight had no major effect on hatchability when injected either on day 5 or day 15 of incubation (hatchability = 90 and 100%, respectively). Plasma, liver and brain esterases were measured in the chick at different time points during incubation and after hatching. Brain acetylcholinesterase (AChE) and liver cholinesterase (ChE) were inhibited significantly during incubation in embryos dosed on day 15 with both carbaryl and aldicarb. Liver carboxylesterase was inhibited significantly during incubation with only the carbaryl treatment. All esterase enzyme activities returned to normal after hatching. Plasma ChE and carboxylesterase levels were not affected with either carbaryl or aldicarb treatment from 8 until 47 days after hatching. Neither carbamate had any effect on brain neuropathy target esterase (NTE) activity either during incubation or after hatching. The locomotion of chicks was affected in both treatment groups until 47 days after hatching. This study indicates that carbaryl and aldicarb may cause long-term delayed alterations in the chicks.     

Toxicity of styrene and styrene oxide on chick embryos

Styrene and styrene oxide were injected into the air space of fertilized chicken eggs at different times during an incubation period of 14 days. The toxicity of styrene and styrene oxide when injected on the fourth day of incubation revealed an LD50 of 40 mumol/egg and 1.5 mumol/egg, respectively. Malformations were found in 0-20% of the embryos, but never in the controls. The results obtained point to a need for further experimental, and possibly epidemiologic, studies on the consequences of styrene exposure.     

Abnormal embryogenesis induced by thiopental

The effects of thiopental on chick embryos were analyzed in the present study. Thiopental was dissolved in saline and injected into embryonating chicken eggs at doses ranging from 0.2 to 4.0 mg per egg. The injections were made into the air sacs of eggs after two to four days of incubation. Control eggs were injected with an equivalent volume of saline (0.1 ml per egg). In all 1080 chicken eggs were used for this study. All embryos were examined on day 7. The LD50 for eggs injected on days 2, 3 and 4 was 2.1, 1.9, and 4.1 mg per egg, respectively. The principal malformations observed were exencephaly, anencephaly, twisted limbs, twisted neck, microphthalmia, everted viscera, and hemorrhage above the left eye and in both cerebral hemispheres. The results of the present study indicate that thiopental has a tendency to cause malformations in the chick embryos tested.     

Potentiating effects of caffeine on the cardiovascular teratogenicity of ephedrine in chick embryos

Ephedrine was administered to 3-day chick embryos (Hamburger-Hamilton developmental stage 19) together with caffeine at doses where each agent alone caused minimal embryotoxicity. Embryos were examined for malformations on day 14 of incubation. The teratogenicity of ephedrine in the chick cardiovascular system was significantly potentiated by caffeine at a dose as low as 0.5 mumol (0.1 mg/egg; 2 mg/kg egg).     

Experimental diabetes model in chick embryos treated with streptozotocin

The aim of the present study was to investigate whether diabetes model can be made by treatment of streptozotocin (STZ) in chick embryos and this model can be used to predict the effect of drug. When STZ (0.3 mg/egg) was injected into the albumen of fertile eggs on the 14th day of incubation, level of blood glucose significantly increased than that of the control on the 17th day of incubation, and level of serum insulin significantly decreased. In addition, the enhanced level of blood glucose in STZ-treated embryos reduced by injection of human insulin. In conclusion, STZ-treated embryos may be applicable to evaluate human insulin and anti-diabetes drugs as an experimental diabetes model.     

Development and validation of a herring gull embryo toxicokinetic model for PCBs

A toxicokinetic model was developed to describe polychlorinated biphenyl (PCB) accumulation by herring gull (Larus argentatus) embryos during development. The model consists of two components, a bioenergetics model that predicts the lipid mass balance of embryo and yolk compartments with time and an empirical toxicokinetic model that describes PCB partitioning between lipid compartments in the egg. The model was calibrated using data on PCB and lipid partitioning between embryo and yolk+albumen at four time points during incubation in herring gull eggs injected with a PCB mixture, combined with data sets on herring gull embryo growth rates and bioenergetic demands with time. The model was validated using independent data consisting of maternally exposed, field-incubated Lake Superior herring gull eggs that varied in incubation ages over the range of 8.5 d to pipping age (26–28 days). PCB concentrations in 6–9 d embryos were nearly an order of magnitude less than predicted by equilibrium lipid partitioning between the embryo and yolk+albumen compartments of the eggs. PCB concentrations in embryos were adequately predicted by equilibrium partitioning, however, for eggs incubated for 23–28 d. An empirical relationship was developed to account for the apparent non-equilibrium behaviour of PCBs during early development. The model was sensitive to the mass of yolk lipids and the mass of PCBs deposited to fresh eggs and much of the variability in embryo PCB concentrations could by explained by accounting for variability in these input parameters. Consistent with experimental data for other avian species, the model predicts that the highest PCB concentrations in the embryo/chick occur during pipping or soon after when yolk lipids have been completely resorbed by the embryo.     

The distribution of 14C-chloramphenicol in the Japanese quail (Coturnix coturnix japonica)

The distribution of 14C-labelled chloramphenicol after oral and intravenous administration to egg laying Japanese quail was studied by whole-body autoradiography. In the liver, kidneys, gizzard, intestinal contents (bile) and oviduct, the 14C-concentration was higher than that of the blood short time after injection and remained higher than the blood up to 4 days. From 4 hrs, the concentration of 14C in the egg yolks was higher than that of the blood and from 24 hrs the radioactivity in the albumen of the eggs in the oviduct was also higher than that of the blood. The peak concentration in the egg yolk was found in the second egg laid 2-4 days after administration of 14C-chloramphenicol. In the albumen the maximum concentration was found in the first laid egg 24-48 hrs after administration. In the egg yolks, about 30% of the radioactivity represented unchanged chloramphenicol up to 5 days after administration. It was also shown that about 5% of the injected 14C-chloramphenicol was exhaled as 14CO2 during the first 12 hrs and about 37% of the dose was excreted in the combined faeces and urine during the same period of time.     

Teratism induced in the developing chick by RPR-V, an organophosphate.

Fertile White Leghorn chicken eggs were injected on day 4 of incubation with 0, 0.25, 0.50 or 1.00 mg RPR-V/egg and opened on day 20 or allowed to hatch. The following parameters were examined: hatchability, growth and development, external malformations and skeletal deformities. As the dose was increased, the hatchability decreased, and the incidence of deformities increased. Staining of the skeleton with Alizarin red showed clear deformities in the embryos. The results suggest that RPR-V has teratogenic effects on chick embryos when injected on day 4 of incubation.     

Time dependent effects produced in chicks after prenatal injection of methylmercury

Methylmercury dicyandiamide (0.05 to 10 mg/kg egg) injected into the yolk sac of fertilized chicken eggs prior to incubation produced a dose related decrease in the percentage of chicks hatched (90-57% of control). With dosage fixed at 0.5 or 5.0 mg/kg egg and injections made on Days 0, 7 or 14 of incubation, hatches were 90, 68 and 75%, respectively, for the low dose and 63, 13 and 18% for the high dose. In contrast to results obtained from chicks hatched from eggs injected on Day 0 of incubation, chicks hatched from eggs injected with 0.5 or 5.0 mg MMD/kg on Day 7 or 14 were not different from controls in a detour learning situation. Administration of 14-C methylmercury revealed maximal brain radiolabel in embryos injected on Day 0 to be 10% that seen with eggs injected on Day 7 but twice that seen with eggs injected on Day 14. We tentatively conclude that a period of maximal sensitivity to the behavior effects exists prior to Day 7 and that the mechanisms of embryolethality is different from that producing the functional deficits.     

Effect of pineal indoles on the chick embryo

In a study on the embryotoxicity of pineal indoles on developing chick embryos in vivo, the pineal indoles--namely, melatonin (MEL), methoxytryptamine (MTA) and methoxytryptophol (MTP)--were injected into the yolk sacs of the chick embryos through the air chambers of the eggs on the 4th day of incubation. The eggs were opened and the embryos examined after 6, 10 or 14/15 days of incubation. Abnormalities were found to occur mainly in the 6- and 10-day-old embryos, which exhibited external malformations such as twisted vertebral column, abdominal hernia, exteriorization of heart and viscera, defects of eye, beak and limb. From the results obtained from embryos on the 14th or 15th day of incubation, MEL was found to be the most toxic indole in regard to the mortality induced, whereas MTA had the highest teratogenicity because of the frequent incidence of abnormal embryos. Effect of MTP treatment on the development of chick embryos varied greatly between doses, and there were no abnormal embryos found on the 14th or 15th day of incubation.     

Cardiotoxicity of trastuzumab (herceptin) in chick embryos

The cardiac toxicity of trastuzumab was studied in chick embryos. Fertilized eggs of White Leghorns were incubated and investigated. Trastuzumab 5 mg/egg (low dose) or 15 mg/egg (high dose) was injected into the air sac of a fertilized egg on the 16th day of incubation. Electrocardiograms (ECGs) were recorded 0 to 60 min after the injection. After low dosing of trastuzumab, the heart rate was not different compared with the control. However, the heart rate was significantly decreased by high dosing of trastuzumab. In addition, arrhythmia was produced by high dosing of trastuzumab. These findings indicate that trastuzumab has a marked dose- and time-dependent influence on the heart rate in chick embryos.     

Aflatoxin deposition and clearance in the eggs of laying hens

Hens fed a diet containing 3310 μg of AFB₁ and 1680 μg of AFB₂ per kg feed for 28 days showed a significant decrease in egg production and egg weights by wk 3 and 4 of feeding, respectively. Transfer of aflatoxins to the eggs occurred rapidly, reaching maximum levels after 4–5 days, and remained relatively constant throughout aflatoxin feeding. The mean values for combined residue levels in eggs were less than 0.5 μg/kg. Levels of AFB₂, AFM₁ and AFM₂ were similar in yolk and albumen while levels of B₁ and B_2a were higher in the yolk. Upon removal of the aflatoxin-containing diet, residues in eggs decreased rapidly. Clearance of aflatoxin residues from the albumen occurred faster than from the yolk. Thus, no residues were detected in the albumen and in the yolk after 5 and 7 days of withdrawal, respectively. No aflatoxin residues could be recovered from whole eggs after feeding the aflatoxin-free diet for 4 days.     

Cardiovascular malformations in the chick embryo induced by thalidomide

The effects of thalidomide on 2- to 4-day-old chick embryos were determined by examining embryos at 7 days of incubation. The LD50 was 3.5 mg, 5 mg and 5.3 mg for developing embryos treated on incubation days 2, 3 and 4, respectively. The cardiovascular anomalies observed were: aortic stenosis (27%), common truncus arteriosus (10.3%), ventricular septal defects (16.2%) and atrial septal defects (7.2%). A considerable damage to the endocardial cushion tissue was found. Aortic arch anomalies, i.e., inhibition of the 3rd and 4th aortic arches separately, and of the 3rd and 4th arches together along with the ventricular septal defects was 11%, 9% and 7%, respectively. No defects were observed in the controls. The results of the present study suggest that thalidomide is markedly toxic for the developing cardiovascular system in the chick embryos.     

Study of danofloxacin depletion in eggs of laying hens after oral administration

Danofloxacin was administered to 15 laying hens via drinking water for 12 days. Egg white and yolk from each egg were separated and danofloxacin residues were analysed by a high performance liquid chromatography method with fluorescence detection. Danofloxacin was detectable on the first day in egg white and on the second day in egg yolk after the beginning of administration, and higher danofloxacin residues accumulated in egg yolk than in egg white. Danofloxacin in egg white decreased fairly rapidly and was detectable up to 4 days after withdrawal of the drug. In egg yolk the residues declined slowly and were detectable up to 11 days after withdrawal of the drug.     

The Distribution of 14C–Chloramphenicol in the Japanese Quail (Coturnix coturnix japonica)

Abstract: The distribution of ¹⁴C–labelled chloramphenicol after oral and intravenous administration to egg laying Japanese quail was studied by whole–body autoradiography. In the liver, kidneys, gizzard, intestinal contents (bile) and oviduct, the ¹⁴C–concentration was higher than that of the blood short time after injection and remained higher than the blood up to 4 days. From 4 hrs, the concentration of ¹⁴C in the egg yolks was higher than that of the blood and from 24 hrs the radioactivity in the albumen of the eggs in the oviduct was also higher than that of the blood. The peak concentration in the egg yolk was found in the second egg laid 2–4 days after administration of ¹⁴C–chloramphenicol. In the albumen the maximum concentration was found in the first laid egg 24–48 hrs after administration. In the egg yolks, about 30% of the radioactivity represented unchanged chloramphenicol up to 5 days after administration. It was also shown that about 5% of the injected ¹⁴C–chloramphenicol was exhaled as ¹⁴CO₂ during the first 12 hrs and about 37% of the dose was excreted in the combined faeces and urine during the same period of time