Elsevier

Environmental Research

Volume 137, February 2015, Pages 185-198
Environmental Research

Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living bird species exposed to different levels of lead under natural conditions

https://doi.org/10.1016/j.envres.2014.12.017Get rights and content

Highlights

  • δALAD ratio may be more sensitive as Pb biomarker than δALAD activity.

  • Blood Pb levels ≥5 µg/dl caused effects on δALAD in Eagle owl.

  • Blood Pb levels ≥8 µg/dl caused effects on δALAD in Griffon vulture.

  • Eagle owls exhibited 79% decrease in δALAD activity when blood Pb exceeded 19 µg/dl.

  • Griffon vultures exhibited 94% decrease in δALAD when blood Pb exceeded 30 µg/dl.

Abstract

The purposes of this study were: (1) to determine the δALAD activity and δALAD ratio in blood of four free-living bird species (Griffon vulture, Eagle owl, Slender-billed gull and Audouin’s gull); (2) and to investigate the correlations between δALAD activity/ratio and Pb concentrations in blood samples. A decrease was observed in δALAD activity in Griffon vultures and Eagle owls exposed to Pb. In addition, negative relationships were found between δALAD ratio or δALAD activity and Log blood Pb levels in Griffon vultures and Eagle owls, and these relationships were stronger in areas with the highest Pb exposure. We provide equations that may be helpful to estimate δALAD activity and δALAD ratio using blood Pb concentrations. Regarding gull species, δALAD activity found in the present study may be considered the normal activity in Slender-billed gull and Audouin’s gull species, since very low blood Pb concentrations and no correlations were found in these species. Although both δALAD activity and δALAD ratio are sensitive biomarkers of Pb exposure and effect in birds, the use of δALAD ratio may improve the results. Besides, this study provides blood threshold concentrations at which Pb bears effects on δALAD enzyme (5 µg/dl in Eagle owl; 8 µg/dl in Griffon vulture; and probably >2 µg/dl in Slender-billed gull and Audouin’s gull). Our findings show that Eagle owl seems to be more sensitive to δALAD enzymatic inhibition by Pb than Griffon vultures. Eagle owls and Griffon vultures exhibited up to 79% and 94% decrease in δALAD activity when blood Pb concentrations exceeded 19 and 30 µg/dl, respectively. Regarding the effects related with δALAD inhibition, significant negative correlations were found between δALAD activity and hematocrit in Eagle owls and Griffon vultures, which may be related to compensatory response associated with a decrease in δALAD activity. In addition, an effect on creatine kinase activity and total proteins in plasma was found in Griffon vultures. The significant negative correlations found between δALAD activity and tGSH in Griffon vulture, and between δALAD activity and Log CAT activity in Eagle owls, may be related to a protective response of antioxidant system against reactive oxygen species (ROS). The negative relationship found between δALAD activity and TBARS levels in Griffon vulture is probably related to an induction of lipid peroxidation by ROS that may be generated by δALA accumulation when δALAD activity is depressed. δALAD activity and δALAD ratio in blood are suggested as important nondestructive biomarkers for Pb exposure and effect for future biomonitoring studies in Griffon vulture and Eagle owl. Further studies are recommended to provide new data on Pb concentrations at which δALAD activity is affected in different wild bird species and to elucidate why different species tolerate Pb in different ways.

Introduction

δ-Aminolevulinic acid dehydratase (δALAD) or porphobilinogen synthase (Enzyme Commission number EC 4.2.1.24) is an essential enzyme in the heme biosynthetic pathway which forms porphobilinogen (PBG) through the condensation of two molecules of δ-aminolevulinic acid (δALA) (Gurer and Ercal, 2000). Lead (Pb) may produce a depression on heme synthesis by the inhibition of three enzymes that catalyze the second, sixth, and final steps, respectively, in the biosynthesis of heme-δALAD, coproporphyrinogen oxidase, and ferrochelatase (Kelada et al., 2001). In this pathway, δALAD is the most sensitive, specific, and well-established biomarker of low Pb exposure and effect in humans and wildlife, including bird species (Pattee and Pain, 2003). This enzyme may be maximally inhibited without other signs of Pb toxicity (Pattee and Pain, 2003). The inactivation of δALAD has been implicated in the pathogenesis of Pb poisoning. In this sense, δALAD inhibition results in a corresponding decrease in heme production and the accumulation of δALA that can be oxidized to generate reactive oxygen species (ROS) (Gurer and Ercal, 2000). Consequently, enhanced lipid peroxidation and DNA damage may occur. Several authors have studied δALAD activity in different avian species exposed to Pb experimentally or under natural conditions (Berglund et al., 2010, Beyer et al., 1988, Beyer et al., 2004, Beyer et al., 2013, Blus et al., 1993, Blus et al., 1995, Carpenter et al., 2003, Gómez-Ramírez et al., 2011, Hoffman et al., 1981, Hoffman et al., 1985, Martinez-Haro et al., 2011, Martínez-López et al., 2004, Vanparys et al., 2008). Nevertheless, there are differences in metal tolerance between different bird species (Beyer et al., 1988, Hernández-García, 2010, Pain, 1987), and information on Pb concentrations, at which δALAD activity is affected in bird species exposed to Pb under natural conditions, is still unclear. Therefore, further studies are needed to understand how the actual environmental Pb exposure affects δALAD activity in free-living birds and the consequences of its inhibition, and to know the threshold concentrations at which Pb may affect δALAD activity and δALAD ratio.

The purposes of this study were to determine the δALAD activity and δALAD ratio in blood of four free-living bird species (Griffon vulture, Gyps fulvus; Eagle owl, Bubo bubo; Slender-billed gull, Chroicocephalus genei; and Audouin’s gull, Ichthyaetus audouinii) exposed to different levels of Pb under natural conditions, and to investigate the correlations between δALAD activity/ratio and Pb concentrations in blood samples. Besides, we evaluated the lowest Pb concentrations at which an effect on δALAD enzyme is produced in four avian species. To our knowledge, this is the first study on the effects of Pb on δALAD enzyme activity in free-living Griffon vulture, Slender-billed gull and Audouin’s gull, and there is only one study on Eagle owl (Gómez-Ramírez et al., 2011).

For Griffon vulture, we selected two different areas that serve as two feeding stations in the Autonomous Community of Valencia (Alcoy, Alicante and Cinctorres, Castellón) (Fig. 1). These two study areas are not considered polluted sites. A previous study involving with the same individuals concluded that Pb levels found in most Griffon vultures could be considered normal or at background levels in vultures feeding all year round on carcasses of pork origin (Espín et al., 2014a). However, three Griffon vultures from Cinctorres had high blood Pb concentrations probably due to ingestion of game meat with bullet fragments in carcasses or with Pb shots embedded in their flesh (Espín et al., 2014a). Judging from the Pb concentrations found in these individuals and the Pb-related oxidative stress found in a previous study (Espín et al., 2014a), we expected to find an effect on the δALAD enzyme activity in Griffon vulture from the Community of Valencia.

As for the Eagle owl, we selected three different areas corresponding to an agricultural and rural area, an industrial area, and an ancient mining site in the province of Murcia (Fig. 1). As it has been recently published in a study on the same individuals (Espín et al., 2014b), Pb concentrations in Eagle owl in the province of Murcia can be considered low. However, local contamination sources have probably contributed to the higher Pb concentrations detected in Eagle owl from the industrial and mining areas comparing with Pb levels in owls from the agricultural and rural area (Espín et al., 2014b). Taking into account the Pb effects previously observed in the same individuals on oxidative stress biomarkes (Espín et al., 2014b), we expected to find an inhibition on δALAD activity in Eagle owls from the ancient mine site in the province of Murcia.

Regarding the Audouin's gull and Slender-billed gull, we selected two studied areas as random locations of interest that have not yet been studied in relation to Pb concentrations and its sublethal effects; the saltworks of Torrevieja for Audouin's gull and the saltworks of San Pedro del Pinatar for Slender-billed gull (Fig. 1). Since the two areas are not considered Pb-polluted, we expected to find low Pb concentrations in gulls and since, to our knowledge, there is no published literature on these Laridae species, that low Pb concentrations may cause inhibition on δALAD activity.

Section snippets

Griffon vulture

The Griffon vulture is a large bird of prey from the Accipitridae family, belonging to the Old World vultures. It is a scavenger that feeds mostly on carcasses of dead domestic livestock and, to a lesser extent, on wild species found dead in the field (Donázar, 1993). The world population of Griffon vultures extends from North Africa, through several South European countries, to Central Asia; and a significant population is concentrated in Spain (Del Moral, 2009). The vulture population has

Blood Pb concentrations, δALAD activity, and δALAD ratio in four free-living bird species

Blood Pb concentrations, δALAD activity, δALAD ratio, and correlations between δALAD ratio and Pb concentrations in Griffon vulture, Eagle owl, Slender-billed gull and Audouin’s gull are presented in Table 1. Out of the four species studied, the Griffon vultures, particularly those from Cinctorres, showed the highest Pb levels (mean±SD=41.4±79.5 µg/dl wet weight, n=30) with significantly higher Pb levels (p<0.01) than those found in individuals from Alcoy (15.3±8.3 µg/dl, n=36) (Table 1) (Espín

Blood Pb concentrations, δALAD activity, and δALAD ratio in four free-living bird species

Pb levels in most Griffon vultures from Alcoy and Cinctorres (median levels of 12.37 and 16.26 μg/dl, respectively) may be normal or background levels in vultures feeding on carcasses of pork origin all year round (Espín et al., 2014a). Although median Pb concentrations were similar among areas, significant higher blood Pb concentrations were found in Griffon vultures from Cinctorres than in individuals from Alcoy (Table 1) (Espín et al., 2014a). These location-related differences are due to

Conclusions

This study provides new data on δALAD activity and δALAD ratios in four different free-living bird species exposed to different levels of Pb under natural conditions. A decrease in δALAD activity was observed in Griffon vultures and Eagle owls exposed to Pb. In addition, negative relationships were found between δALAD ratio and blood Pb levels in free-living Griffon vultures and Eagle owls, and these relationships were stronger in the areas with the highest Pb concentrations. Regarding gull

Acknowledgments

The authors thank MICIIN for financial support through projects CGL2008-4318/BOS and NOVEDAR-Consolider, and Seneca Foundation for project 08758/PI/08. Thanks also the European Science Foundation for EURAPMON network. Special thanks to the Wildlife Service and to the Protected Areas Management Service, both belonging to the General Directorate of Natural Environment from the Conselleria de Infraestructure, Territori I Medi Ambient (Generalitat Valenciana) for the permissions and collaboration

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    Present address: Section of Ecology, Department of Biology, University of Turku, 20014 Turku, Finland. silvia.

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