Comparative Biochemistry and Physiology Part C 124 (1999) 197 – 202 www.elsevier.com/locate/cbpc
Developmental changes in 7-ethoxyresorufin-O-deethylase (EROD) and d-aminolevulinic acid dehydratase (ALA-D) activities in three passerines Susanna Tanhuanpa¨a¨ a,*, Tapio Eeva b, Esa Lehikoinen b, Mikko Nikinmaa a a
Department of Biology, Laboratory of Animal Physiology, Uni6ersity of Turku, FIN-20014 Turku, Finland b Department of Biology, Section of Ecology, Uni6ersity of Turku, FIN-20014 Turku, Finland Received 1 April 1999; received in revised form 16 July 1999; accepted 19 July 1999
Abstract In this study, we report changes in 7-ethoxyresorufin-O-deethylase (EROD) and d-aminolevulinic acid dehydratase (ALA-D) enzyme activities during development of three wild passerine bird species: pied flycather (Ficedula hypoleuca), great tit (Parus major) and blue tit (P. caeruleus). Activities were determined from late embryos, newly hatched, 1-week-old and 15-day-old nestlings and adult birds. EROD activity from hepatic microsomes and ALA-D activity from liver tissue and erythrocytes were measured. In all species investigated EROD and ALA-D activities were increased during the first posthatching week and decreased thereafter. Also, differences between activities in late-term nestlings and adults were seen. Based on these results, we suggest that the developmental status of an animal must always be taken into account when evaluating the results obtained in the biomarker studies. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Passerines; Ficedula hypoleuca; Parus major; Parus caeruleus; EROD; ALA-D; Basal activity; Ontogeny
1. Introduction 7-Ethoxyresorufin-O-deethylase (EROD), a member of a cytochrome P450IAI (CYP1A1) enzyme family, has been used as a biomarker in detecting exposure to, e.g. polyhalogenated aromatic hydrocarbon compounds in birds [18]. Cytochrome P450 enzymes, also called mixed function oxidases, are involved in metabolizing of both exogenous toxicants and endogenous compounds, such as steroid hormones and fatty acids to more polar compounds which can be excreted directly or after conjugation with water-soluble agents in phase II reactions [12,17,20]. Usually this reduces the toxicity of the compounds, but in some cases they are converted to products with greater toxicity. d-Aminolevulinic acid dehydratase (ALA-D) participates in the heme biosynthesis by catalysing the condensation of two aminolevulinic acid (ALA) molecules * Corresponding author. Tel.: +358-2-333-5785; fax: + 358-2-3335953. E-mail address:
[email protected] (S. Tanhuanpa¨a¨)
to form porphobilinogen (PBG), the precursor for porphyrins, and its activity is inhibited by lead [18]. Inhibition of ALA-D activity has become a standard method in diagnosis for lead intoxication in humans, and it has also been commonly used as a biomarker in detecting lead exposure in animals, including birds. In an avian in vitro system lead has been found to be a 10–100-fold more potent inhibitor for ALA-D activity than copper, cadmium or mercury [25]. ALA-D inhibition is evident within 4 weeks after ingestion of one lead shot pellet [6] and more quickly in birds with daily lead doses [8,11]. Inhibition is still detectable at least 3 months after the cessation of the exposure [6]. Although EROD induction and ALA-D inhibition have been frequently used as biomarkers of environmental contamination in birds, little is known about their basal activity levels during development, especially in wild bird species. Yet, age is one of the important factors affecting the activity of these enzymes [18]. In this study, we report basic activity levels for EROD and ALA-D enzymes during the ontogeny in three small passerines. Species used were pied flycatcher (Ficedula
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hypoleuca), great tit (Parus major) and blue tit (P. caeruleus). We present EROD activities in hepatic microsomes and ALA-D activities in erythrocytes as well as in hepatic tissue. Developmental stages investigated were late embryos (approximately 2 days before hatching), newly hatched, 7-day-old and 15-day-old nestlings. Data are also presented for adults with an age of at least 1 year.
2. Materials and methods
2.1. Sample collection Blood and liver samples from 79 1- and 159 2-day old and adult birds were collected at three study sites with no major pollution sources in south-west Finland. For each developmental stage, we took samples from six to seven birds living in randomly chosen nests. Laying and hatching dates were determined by visiting the nests regularly. To get data on embryos and newly hatched birds, eggs were collected from nests in the same study areas and incubated in the laboratory at 40°C in a humidified atmosphere. Eggs were turned every 6 h. Samples were taken from embryos approximately 2 days prior hatching. Due to the difficulties in determining the sex of young birds, the nestlings used were of mixed sex. All the adult birds examined were females trapped at the nest. For sampling the birds were killed by decapitation. Blood samples for ALA-D determinations were taken from the jugular vein with heparinized 25 or 50 ml micro capillary tube and mixed with four volumes of 1% saponin/0.1% Triton X-100 in 1.5 ml Eppendorf tubes. After vigorous shaking, hemolysates were stored in liquid nitrogen. Livers were removed immediately after blood sampling and stored in liquid nitrogen. In order to get enough material for enzyme determinations, liver samples from four to six embryos and 0-day-old hatchlings were pooled.
2.2. ALA-D acti6ity ALA-D activities in erythrocytes were determined from newly hatched nestlings to adults. Values from hepatic samples were obtained from late embryos until adults except in F. hypoleuca, where liver samples for the ALA-D assay were not available in birds younger than 7 days. The method used was modified for birds according to Scheuhammer [25]. For hepatic samples activity was measured using the whole cell fraction prepared by homogenizing livers in ice cold 0.9% NaCl, and then centrifuging the homogenate at 8000× g for 15 min at 4°C to remove cell debris. For activity measurements, a 50 ml sample (blood hemolysate in 1% saponin/0.1% Triton X-100 or hepatic whole cell frac-
tion) was added to a mixture containing 50 ml of 0.5 M morpholinethanesulfonic acid (MES) buffer (pH 6.6), 25 ml of distilled water and 25 ml of 60 mM 5aminolevulinic acid hydrochloride (ALA; Merck). Samples were incubated for 1 h at 42°C and the reaction was stopped by adding 100 ml of 0.4 M trichloroacetic acid (TCA)/60 mM Hg2 + . After centrifugation at 15 000×g for 10 min, one volume of the resulting supernatant was added to 7.5 volumes of modified Erlich’s reagent. After 10 min, the absorbance was read at 555 nm with a Perkin Elmer Lambda Bio UV/Vis Spectrometer. All the enzyme assays were performed as duplicates. The activity was calculated as picomoles of porphobilinogen (PBG) formed per hour per milligram of protein using 6 × 104 as the molar extinction coefficient for PBG [13]. Protein concentrations were determined by the method of Bradford [2] using the BioRad Protein Assay Kit with bovine serum albumin as a standard.
2.3. EROD acti6ity EROD activity was measured using liver microsomes which were prepared in the following fashion. Liver samples were rinsed with ice cold 1.15% KCl and homogenized in four volumes of 0.25 M sucrose. The homogenate was centrifuged at 8000× g for 20 min at 4°C and the resulting supernatant was further ultracentrifuged at 100 000×g for 60 min at 4°C. The microsome pellet obtained from ultracentrifugation was resuspended in one volume of SET buffer at 4°C (0.25 M sucrose, 5.4 M EDTA, 66 mM Tris–HCl; pH 7.4 at 20°C). Activity was determined fluorimetrically according to the method described by Burke and Mayer [3], with minor modifications. A reaction mixture contained 65 mM Tris–HCl buffer (pH 7.4), 1 mM 7-ethoxyresorufin (Sigma) and 30 ml of sample in a final volume of 1962 ml. The reaction was started by adding 40 ml of 50 mM NADPH (b-nicotinamide adenine dinucleotide phosphate; Sigma). Samples were excited at 510 nm and the emission was measured at 586 nm at 42°C with a Perkin Elmer Luminescence Spectrometer LS 50. Resorufin (Sigma) was added at a 50 mM final concentration as an internal standard and the activity was calculated as picomoles resorufin formed per minute per milligram of microsomal protein. Each sample was measured as duplicates. Protein concentrations were determined as described above.
2.4. Statistics The statistical significance of differences in enzyme activities between age groups and between species was determined by using a one-way analysis of variance (ANOVA). The level of significance was set at 0.05 and values in figures are given as 9 S.E.
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3. Results
3.1. ALA-D acti6ity ALA-D activities were measured in blood (Fig. 1) and in hepatic tissue (Fig. 2). In erythrocytes, there was a general trend that activities increased from the hatch until 1 week of age and decreased thereafter with increasing age. In the liver, there was also an increase in activities during the first posthatching week in P. major and P.caeruleus nestlings. However, the peak observed at day 7 was more pronounced in the liver than in erythrocytes. Due to the difficulties in obtaining enough liver tissue from F. hypoleuca, no data is available for birds younger than 7 days. We did not find any change in activity in F. hypoleuca between 7 and 15 days of age. However, the values in nestlings were much higher than those of adults. In P. caeruleus, liver ALA-D
Fig. 2. Developmental changes in liver ALA-D activity (pmol/h/mg) from late embryos to adults. For birds aged 7 days or older, each point represents a mean ( 9S.E.) for six to seven individuals. In late embryos and day 0 hatchlings data is presented for one to two pooled samples (n = 4 – 6 livers/pool). In F. hypoleuca no data is available for birds younger than 7 days. * Significantly different (PB 0.05) from previous age group. a Significantly (P B0.05) different from P. caeruleus. b Significantly different (PB 0.05) from P. caeruleus and P. major.
activities were higher in the adults than in 15-day-old nestlings, but in P. major no difference was found between these groups.
3.2. EROD acti6ity
Fig. 1. Developmental changes in erythrocyte ALA-D activity (pmol/ h/mg) from newly hatched birds to adults. For birds aged 7 days or older, each point represents a mean ( 9 S.E.) for six to seven individuals. In newly hatched birds data is presented as a mean (9S.E.) for two to 14 individuals. * Significantly different (PB0.05) from previous age group. a Significantly different (PB0.05) from P. caeruleus. b Significantly different (P=0.05) from P. caeruleus and P. major.
EROD activities measured for hepatic microsomes of birds in different ages are shown in Fig. 3. There was an increase in activity during the first posthatching week in all three study species. In F. hypoleuca, however, the increase was more pronounced (over a 100fold) than in the other species. After an increase at 7 days there was a significant decrease in activity in all species. In F. hypoleuca, the activity was higher in adults than in 15-day-old nestlings and the adult values were comparable to those of 7-day-old nestlings. In P.
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major and P. caeruleus no significant differences between 15-day-old nestlings and adults were found. The values for F. hypoleuca were significantly higher than those measured for the other species for 7- and 15-day old nestlings and adult birds.
4. Discussion In this study, we examined the ontogenic changes in the activities of two enzyme systems, 7-ethoxyresorufinO-deethylase (EROD) and d-aminolevulinic acid dehydratase (ALA-D), which are both commonly used as biomarkers in monitoring environmental contamination. The determinations were performed in three small passerine bird species, pied flycatcher (F. hypoleuca), great tit (P. major) and in blue tit (P. caeruleus). Activities were measured from late embryos to adults.
Fig. 3. Developmental changes in hepatic microsomal EROD activity (pmol/min/mg) from late embryos to adults. For birds aged 7 days or older, each point represents a mean for six to seven individuals. In late embryos and newly hatched birds data is shown for one pooled sample (n = 4 – 6 livers/pool). * Significantly (PB 0.05) different from previous age. b Significantly (PB 0.05) different from P. caeruleus and P. major.
EROD activity was low at hatch in all study species and peaked at 7 days posthatch. These results suggest that a peak in EROD activity occurs later than in chicken (Gallus domesticus), where mixed function oxidase activities are high already around hatching and reached maximum at 1 day of age [10,16,21]. However, passerine nestlings are altricial and the overall developmental status of the altricial hatchlings is known in many respects to correspond with mid -to late-stage embryos of precocial species. Therefore, the observed delay in reaching the peak EROD activity in our study species compared to chicken may be related to the developmental differences. In wild bird species, only a few studies are available so far concerning the ontogenic development of the cytochrome P450 enzyme activities. However, our results are consistent with studies made in other altricial species. Sanderson et al. [24], found that testosterone hydroxylase activities were consistently lower in great blue heron (Ardea herodias) hatchlings than in older birds. Similarly, Rinzky and Perry [23] measured significantly lower cytochrome P450 protein content in the hepatic tissue of barn owls (Tyto alba) at day 1 than at other ages. It should be kept in mind, however, that cytochrome P450 protein content does not always correlate well with the catalytic activity, especially during early development [26,27]. In herring gull (Large argentatus) Peakall et al. [19] found similar trends in the development of EROD activity as what is reported in this study. Their data also suggest that different forms of CYP may have different time courses. It is reported in mammals that mixed function oxidase activities are generally low at birth and increase thereafter during development [9,14,15]. At present, we cannot say exactly when the highest EROD activity is reached during development in our study species, since only newly hatched and 7-day-old-birds were sampled. More detailed studies are needed to determine the very early development of cytochrome P450 activities in these three passerines. The factors affecting the early induction of the cytochrome P450 system may be several. Residues of the yolk still remaining in the egg is resorbed into the embryo just before hatching, and it has been hypothesized that the increase in cytochrome P450 activities observed after the hatch might be due to the metabolism of the lipid soluble components of the yolk [21]. In addition, after the hatch nestlings are fed for the first time with animal food which is not predigested and metabolized. The period from hatching to 1 week of age is the most rapid growth stage of small passerines [22]. Therefore, the observed increase in activity of EROD may be due to the overall high metabolic rate and there are probably also hormonal changes associated with this developmental point. Taken together, it seems that the early EROD induction reflects the im-
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portance of the cytochrome P450 system in metabolism of endogenous compounds as well as its participation in hormone synthesis during the early stages of development. Also, it is likely that after hatching the nestlings, metabolism will encounter many new chemicals which it has not had to deal with in ovo and thereby inducing the mixed function oxidase system. In F. hypoleuca, a second increase in EROD activity between 15-day-old nestlings and adults was seen. Rinzky and Perry [23] found a change in hepatic cytochrome P450 protein level in barn owls between 30 and 40 days of age, when they changed diet from concentrated food provided by the mother to regular food. Also in rats, weaning was found to cause changes to the activities of cytochrome P450 enzymes [1,27]. Interestingly, of the species used, only F. hypoleuca is migratory, and thus, the EROD activity may still be influenced by the food consumed at the wintering site and by the migratory route. However, studies are required for the variations in the EROD activity of this (and other) migratory species and resident species of different locations. ALA-D enzyme plays a critical role in the heme biosynthetic pathway. Thus, one would expect an increase in ALA-D activity with an increase in erythropoietic activity. Indeed, a direct, linear correlation between ALA-D activity and blood reticulocyte percent has been observed in rat [5]. In birds, there is a transition to pulmonary respiration from passive diffusion at the time of hatching. In the fledging state, chicks are faced with the high oxygen demand of flight. Consistent with these facts we observed an increase in both erythrocyte and hepatic ALA-D activity during the first 7 days after hatching in all study species. In erythrocytes the activity was still significantly higher in 15-day-old nestlings than in adults. Similarly, Grue et al. [8] found in barn swallow (Hirundo rustica) that ALA-D activities were approximately two times higher in 16 – 18-dayold nestlings than in adults. However, erythrocyte haemoglobin content was significantly lower in nestlings compared to adults indicating that the haematopoietic system is still under development in this stage. Hoffman et al. [11] measured the basal ALA-D activity levels in developing American kestrel (Falco spar6erius) red blood cells and in brain and liver tissues. During the first ten posthatching days they reported a nearly 4-fold increase in brain ALA-D activity and also a marked increase in hepatic activity. In erythrocytes, however, they detected a nearly 30% decrease during the same time period. In mammals the activities are generally found to be high in young animals and decrease with increasing age. In calves, for example, the activity increased from 1 to 9 weeks of age, before it slowly returned to the initial values [4]. The information concerning the ontogenic development of the ALA-D activity is, however, still limited even in mammals.
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In our earlier study [7] we did not see ALA-D inhibition in passerine nestlings although lead contamination was evident in the study area. The data from the current study supports our previous suggestion that the rapid development of the hematopoietic system in young nestlings can interfere with the inhibition of ALA-D by lead. In conclusion, we report that in the small passerine bird species investigated the activities of EROD and ALA-D enzymes increased during the first posthatching week and decreased thereafter. Also, late-term nestlings had much higher erythrocyte ALA-D activities than adults in all three species, and in the flycatcher much lower EROD activity than adults. Owing to these differences, the developmental stage of an animal must be taken into account when evaluating the results obtained from the biomarker studies.
Acknowledgements We are most grateful to Jorma Nurmi and Antti Tuominen, who helped us in sample collection. This study is financially supported by the Academy of Finland.
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