Delta-aminolevulinic acid dehydratase (ALAD) characterization and use in metal exposure assessments

Environmental contamination as a result of anthropogenic activities has greatly increased the global availability and distribution of potentially toxic metals. Of these metals, the use of lead (Pb) by humans for millennia has resulted in Pb poisoning achieving the unfavorable distinction as the most common disease of toxic environmental origin in the United States today. Solely as a result of man’s actions, Pb has become the most widely dispersed toxic metal in the world. Methods intended to measure exposure and adverse effects of Pb have assessed the ability of living organisms to bioaccumulate the metal and to respond physiologically. Lead is a multitargeted toxicant, with hematotoxicity a primary effect, specifically inhibition of heme synthesis. ALAD is an enzyme of the heme synthesis pathway, whose inhibition in humans and animals has served as an important, and sensitive, biological marker for Pb exposure and injury. Blood samples from mammal, bird and reptile species were characterized for ALAD activity, median inhibitory Pb concentrations (IC50) and reactivation in Pb inhibited ALAD as a result of dilution in deer mice, Sprague-Dawley rats, American kestrels, prairie voles, European starlings, western diamondback rattlesnakes, Morelet’s crocodiles, Texas horned lizards and domestic pigs. Five avian and two small mammal species were collected from the Anaconda Smelter Superfund site (Deer Lodge County, MT) and assessed for ALAD activity as a biomarker of exposure to smelter-related metal contamination. Literature reviews describe blood ALAD response to environmental Pb contamination or from Pb dosing experiments in wild, captive or laboratory small mammals and birds. Results of ALAD assay development in multiple species described enzyme accuracy, hemolysate preparation, buffer selection, effect of changes in pH, blood dilution and incubation time on enzyme activity, analysis of kinetic parameters (Vmax, Km), IC50 determinations and blood dilution effects on reactivation of Pb-inhibited ALAD activity.

Maximal enzyme activity was observed at pH 6.4 in all animals studied except the Texas horned lizard (pH 5.8). Dilution of blood in the American kestrel (60 to 120:1), domestic pig (30 to 120:1), prairie vole (7.5 to 120:1), deer mouse (15 to 60:1) and rat (7.5 to 30:1) demonstrated species-specific linearity of ALAD activity. Blood Vmax values varied from 7.96 to 78.95 nmol ALA/min*ml RBC in the Morelet’s crocodile and American kestrel, respectively, while Km ranged from 46.5 to 643.1 uM in the Morelet’s crocodile and Texas horned lizard, respectively. There was little difference in enzyme sensitivity (10 min IC50, µM) to in vitro Pb exposure when assessed in test species: domestic pig (1.10 ± 0.25 µM) > American kestrel (1.84 ± 0.53 µM) > deer mouse (6.27 ± 1.21 µM) > European starling (6.73 ± 0.98 uM). Reactivation of Pb-inhibited ALAD activity as a result of blood sample dilution in deer mice, Sprague-Dawley rats and domestic pigs demonstrated recovery of activity from 10 to 55%, 8 to 66% and 58 to 89%, respectively. Reactivation of enzyme activity as a result of the assay procedure suggests that an underestimation of injury to biological resources may occur when ALAD is used in assessments as defined by federal statute. Of the animals collected from the Anaconda Smelter site and assessed for blood ALAD activity, the northern pocket gopher had the most complete data to determine relationships among exposure to soil metals, tissue residues and ALAD activity.

Significant relationships were determined between soil and blood Pb, soil Pb and ALAD, blood Pb and ALAD, and kidney Pb and ALAD in the pocket gopher. The interactive effects of blood Zn and Pb on ALAD activity were demonstrated using a multiple regression model and the ratio of concentrations of blood Pb and Zn (Pb/Zn). In the deer mouse, blood ALAD displayed no relationship to soil, blood or kidney Pb concentrations. Meadow vole ALAD activity was not correlated with variations in concentrations of soil Pb. Though trends were evident, American kestrel nestlings displayed non-significant relationships with blood Pb or blood Pb/Zn ratio, and ALAD activity. In the European starling, blood and kidney Pb and the blood Pb/Zn ratio significantly predicted variations in ALAD activity. Mountain bluebird blood and liver Pb and blood Pb/Zn ratio were significantly related to ALAD. In the three avian species, there was an apparent threshold for ALAD inhibition at a blood Pb concentration of 0.11 ug/g. The interaction effects of Zn and Pb on ALAD activity have been well characterized and are clear in their relative effects on the enzyme. The mechanism of ALAD inhibition is the replacement of the enzyme-bound Zn by Pb. The interaction of these metals on enzyme function is demonstrated in regression plots with the variable expression of ALAD activity under varying concentrations of blood Pb and Zn. An examination of the unusual lack of ALAD response to blood Pb concentrations in wild-collected deer mice from the Anaconda Smelter Superfund site was conducted using a laboratory-raised, wild-type conspecific (P. m. bairdii) exposed to Pb and concurrent Cu or Zn supplemented drinking water. Deer mice were dosed for 21 days with Pb acetate at 0, 0.1, 1, 10, 100, and 1000 ug/ml in drinking water that corresponded to total daily intake of Pb of approximately 0.05, 0.08, 0.4, 3, 30 and 300 mg/kg body weight. Significant elevations of tissue Pb above control were seen in the 10, 100 and 1000 ug/ml groups in blood (0.025, 0.19, 0.66 ìg Pb/g blood, respectively), and 100 and 1000 groups in liver (1.0 and 6.1 ug Pb/g liver wet wt., respectively) and kidney tissues (3.2 and 25 ug Pb/g kidney w.w., respectively). Blood ALAD activity was inhibited approximately 20, 50, and 80% at the three highest doses, consistent with findings in standard laboratory rodent species.

Results of exposure to the micronutrients Zn and Cu, concurrent with effect- inducing Pb levels, were evaluated on Pb accumulation and ALAD activity. Deer mice were dosed for 28 days with Pb acetate at 0 and 100 µg/ml as well as increasing concurrent concentrations of either Cu (10 to 100 µg/ml) or Zn (30 to 300 µg/ml) in drinking water. The higher doses of both Cu and Zn led to a decreased accumulation of Pb. Zinc significantly reduced blood Pb accumulation by over 55% in the highest dose group. In addition, Zn protected against Pb-induced ALAD inhibition by up to 60% in the highest dose group when compared to the positive Pb control. The wide spread use and distribution of environmental Pb has negatively impacted many living organisms by inducing lethal and sublethal effects. As a sensitive biomarker of Pb exposure, the ALAD enzyme was characterized and applied in the assessment of Pb-induced toxicity in controlled laboratory small mammal studies and in wild-collected species. ALAD was responsive through a wide range of Pb exposures, from no effect levels, through levels leading to frank pathology in the kidney. ALAD activity is an appropriate measure of direct physiological response to Pb and was found to be applicable in new wildlife species under field conditions of Pb exposure.