Serum Protein Profile and Blood Cell Counts in Adult Toads Bufo Arenarum (Amphibia: Anura: Bufonidae): Effects of Sublethal Lead Acetate

. Lead is a multiple-source pollutant, well known for its toxicity, of great risk both for the environment and human health. The main target organs of lead are the hematopoietic, nervous, and renal systems; there are also reports in support of its impairment effects on the reproductive and immune systems. It is well known that most of the metal is accumulated in the blood cells and that many of the deleterious effects are related to its circulating concentrations. These adverse effects have been described not only in humans but also in a number of other vertebrates such as fish and birds. The purpose of the present work was to evaluate the effects of weekly administration of sublethal Pb (as acetate, 50 mg . kg ) 1 ) during 6 weeks on the profile of the serum proteins and blood cell counts of the adult South American toad, Bufo arenarum (Anura: Bufonidae). The electrophoretic patterns of serum proteins pointed out the presence of four fractions; the metal provoked a significant decrease in both total proteins and albumin fraction; among the globulin fractions, the G3 resulted augmented. These findings may be related to the impact of lead on the toads ’ hepatic cells and immune system. The number of total red blood cells (RBC) showed a tendency to decrease after the injections of the metal, whereas the

Heavy metals are ubiquitous in the biosphere, where they occur as part of the natural background of chemicals.Anthropogenic activities have introduced substantial amounts of them into the environment by mobilization from their natural insoluble deposits or environmental sinks.Thus, there has been a substantial change in the natural pattern of location and deposition, magnifying the distribution of heavy metals in the ecosystems which, in turn, has provoked a sustained increase in the exposure of the biota to their toxicity (Newman and Unger 2003;Fink and Salibiµn 2005).
Among those heavy metals, lead (Pb) is a multiple-source pollutant, a well-known toxic element of great risk both to the environment and to human health (WHO 1977(WHO , 1989(WHO , 1995)), and is recognized as a carcinogen in experimental animals, and classified as a possible carcinogen in humans (IARC 1987;Jemal et al. 2002).It has no known essential role.It is well documented that its accumulation in tissues induces a broad range of essential physiological, biochemical, and behavioral dysfunctions in a dose-related fashion, Hence, Pb is a major issue in environmental health.In fact, the present level of Pb in blood of nonexposed individuals living in Buenos Aires City, Argentina, is between 125 and 1000 times higher than that of preindustrial populations, which can be equivalent to unexposed humans (Silbergeld 1995;Tong et al. 2000).
Most of the metal is accumulated in the blood cells, and many of the toxic effects correlate with its circulating concentrations (WHO 1995).The main target organs for the action of Pb are liver, hematopoietic and nervous systems, and kidney (Goering and Fisher 1995;Woods 1995;Daniell et al. 1997); there are reports in support of its deleterious effects on the reproductive and immune systems of humans (Mishra et al. 2003).
Those detrimental effects have been described not only in humans but also in a number of vertebrates such as fish, amphibians, birds, and nonhuman mammals during exposure to sublethal concentrations; the observed effects can persist after removal of the contaminant.
The ecological position of amphibians is fundamental in the trophic networks of the ecosystems.The damage that can take place in the population of amphibians as a consequence of its exposure to heavy metals, even at sublethal concentrations, leads to important and widespread ecological consequences (Sarkar 1996), provoking very different events, which can trigger a cascade of adverse secondary effects tending to affect species from other levels, Thus, reduction of amphibian populations could mean substantial direct or indirect changes in other populations trophically dependent on them.Several authors have reported evidence showing that a large number of amphibian populations are declining worldwide (Mann and Bidwell 1999;Houlahan et al. 2000), including Latin America (Lips et al. 2000;Ron and Merino 2000).A number of natural and anthropogenic causes have been suggested to explain this complex phenomenon, mentioning the sustained increase of heavy metals in the environment.
Concerning anuran amphibians, several studies carried out in our laboratory revealed information about the impact of sublethal doses of pb on different functions of target organs of adult specimens of the South American toad Bufo arenarum (Perí et al. 1998a(Perí et al. , 1998b;;Rosenberg et al. 1998Rosenberg et al. , 2000Rosenberg et al. , 2002Rosenberg et al. , 2003;;Arrieta et al. 2000aArrieta et al. , 2004;;Rosenberg 2001).
The purpose of the present study was to evaluate the effects of long-term administration of sublethal Pb (as acetate) on the profile of the serum proteins and blood cell counts of adult B. arenarum.A preliminary report of the results was published elsewhere (Chiesa et al. 1999).

Animals
Forty-eight adult B. arenarum male specimens (average body weight 120 g) were collected from the surroundings of La Plata City, Argentina.The animals were housed individually in plastic containers with tap water, and were preadapted in a chamber at constant photoperiod and temperature (12 D:12 N, 20°C) for 7 days; water was renewed once a day; the toads continued staying in the same environmental conditions throughout the experiments, and were fed minced bovine meat once a week.

Lead Administration
Two solutions, containing Pb acetate and Na acetate, respectively, were prepared in distilled water.The group of experimental toads (n = 22) received one injection of Pb acetate every week for 42 days, which equals a dose of 50 mg Pb 2+ .kg )1 ; the control toads (n = 26) were simultaneously injected with Na acetate solutions.The injections were performed in the dorsal lymph sac, at a rate of 0.6 ml .100 g )1 body weight.The weekly injected dose of Pb was previously determined in our laboratory as sublethal at 20°C for toads of comparable mean body mass; it corresponded to 5.6% of the 120 h-LD 50 (Arrieta et al. 2000b).No mortality was recorded either in Pb-injected toads during the experimental period.

Blood Collection: Total and Differential Leukocyte Counts
Blood samples were collected from toads anesthetized with MS-222.The first sample was taken 3 days before the first injection and the second one was taken 7 days after the last injection.In the last case, toads were also pithed and blood was then obtained by heart puncture.In all cases, samples were collected on EDTA .K 2 .
Cell counts were performed on samples taken from two subgroups of 12 toads each (control and Pb-injected) randomly selected.They were made in duplicate in a Neubauer chamber, in whole blood diluted 1/200 in amphibian Ringer solution.Different morphological typing of the cells was done following the description made by Varela and SellarØs (1937).Red blood cell (RBC) counts were expressed as number of cells • 10 8 .ml )1 , as mean € SD.Differential leukocyte counts were determined in blood smears stained with May Grunwald-Giemsa solutions.Results were expressed as absolute (• 10 7 .ml )1 ) and relative percentages obtained from a count of 100 cells, as mean € SD.

Blood Lead Concentrations
The whole blood Pb concentration was determined by atomic absorption spectrometry in a Varian SpectrAA model 300 spectrometer (Varian, Lexington, MA).Aliquots of blood samples were digested with concentrated HNO 3 in a water-bath at 70°C, and were filtered through Whatman No. 1 and nitrocellulose MSI 0.45 discs.The calibration curve was carried out adding Pb nitrate solutions to whole blood control samples with the same matrix as the treated ones, following the specifications of the APHA-AWWA-WPCF (Clesceri et al., 1998).The detection limit was 0.1 mg .dl )1 .The linear regression for the calibration curve was y = )0.006+ 0.0575 • (r = 0.993, p = 0.007).All reagents were analytical grade.Blood Pb concentration was expressed in mg .dl )1 , as mean € SD.

Serum Protein Concentrations and Fractionation
Sera were separated by centrifugation of whole blood at 600 g.The total serum protein determination was made with the Biuret method using a commercial kit.An electrophoretic separation on cellulose acetate (TITAN III) was performed to determinate the concentration of the protein fractions.The buffer employed was Tris-Barbital-Barbital Na, pH 8.8.The running time was 15 minutes at 200 V. Human serum was used as methodological control.The cellulose strips were stained with 0.5% Ponceau-S (in aqueous 3,5% (w/v) sulfosalicylic acid solutions and 3.5% (w/v) trichloroacetic acid), and were read in a Helena Auto fluor Scanner Flur-Vis densitometer (Helena Biosciences, Collierville, TN).
Four zones were separated, and they were denominated albumin and globulins G1, G2, and G3, after Bertini and Cei (1960).
Results were expressed as g .dl )1 mean € SD.All employed reagents were analytical grade.

Statistical Analysis
Student's t test the correlation tests and analysis of variance (ANOVA) were carried out using the StatgraphicsPlus statistical package (Manugistics Inc., Rockville, MD).The assumption of normality and homogeneity of the variance were previously verified.Significance levels were fixed in 5%.

Results
Blood Pb concentrations at the beginning and at the end of the experimental period, both in control and in lead-injected toads, are shown in Table 1.
In control toads, a considerable concentration of Pb was determined.We reported similar findings in previous studies (Rosenberg et al. 2003;Arrieta et al. 2004).At the end of the experimental period, the whole blood Pb concentration in this group of animals showed a significant reduction (approximately 30%).On the other hand, the blood of treated toads showed a highly significant almost threefold increase in its Pb concentration at the end of the same period.Similar behavior was also reported in another set of experiments (Arrieta et al. 2000a).
When comparing the Pb levels registered in the blood of both groups of toads at the end of the 6-week experimental period, a four fold increase was found.This increase in blood Pb represented 1.1% of the total amount of Pb given at the end of the 6-week period (36 mg for a mean body weight of 120 g); that percentage came out considering a blood volume of 7 ml (Rouf 1969;Carmena-Suero 1980) and the increase in Pb concentration from 2.9 to 8.6 mg .dl )1 .
Tables 2 and 3 show the values of the total RBC and white blood cells (WBC) and differential leukocyte blood counts at the end of the experiments.It can be seen that in Pb injected toads the number of RBC decreased nonsignificantly (by 34%), whereas the number of WBC and blast-like cells was significantly increased.
The results related to the effects of Pb on serum proteins are presented in Figure 1 and Table 4.In Figure 1, electrophoretic patterns of B. arenarum and human sera are comparatively shown.Contrary to what is observed in humans, sera of toads were fractioned to only four zones that were separated; albumin, G1, G2, and G3 in growing order of molecular weight.The mobility of the toads' albumin fraction was similar to the same human fraction; however, the mobility of the globulins and of the remaining fractions was higher than in humans, which might be attributed to differences in molecular weights as well as in electric charges.
Table 4 shows the results of the concentrations of total serum proteins and their fractions.Comparisons were made between control and Pb-exposed toads, and between the initial and final time in each group under study.In the control group, there were no statistically significant differences between initial and final time, suggesting that the captivity condition of the toads did not affect their blood protein concentration.In the Pb-injected group of toads, the only statistically significant increase was noted in the G3 fraction concentration.When comparing the values determined in both groups of toads at the end of the experimental period, a significant decrease in the total proteins was found.In analyzing the electrophoretic fractions, a decrease in the albumin fraction was also found.On the other hand, the G3 fraction was significantly augmented Besides, in the Pb-injected group, the level of this fraction was higher than the level at the starting time.

Discussion
Although many sources of Pb have recently been eliminated, excessive Pb-exposure is still a major environmental issue in Latin America (García-Fernandez et al. 1990;Albert and Badillo 1991;Romieu et al. 1997;MaÇay et al., 1999;Flores and Albert 2004;Paoliello and De Capitani 2005).Several authors reported the presence of Pb in blood in nonexposed humans (Cµrdenas et al. 1993;Calderón-Salinas 1996;Bergdahl et al. 1998;Lopez et al. 2000).Similar findings of wild or noninjected animals, but showing measurable metal concentrations, were reported by other authors (Stansley and Roscoe 1996;Bergdahl et al. 1998;Berzins and Bundy 2002).Surprisingly, in some invertebrates an increase in the performance of some physiological processes after exposure to elevated environmental amounts of Pb was reported (Beeby and Richmond 2001).
Evidence of that situation is the fact that both the blood of wild specimens of toads (Arrieta et al. 2001) and our noninjected control toads contained some amount of the metal in their blood; in addition, the blood Pb recorded at the beginning of the experimental period in control toads did change after 42 days, showing a significant decrease.We had previously shown that keeping specimens of the same species for shorter periods of time in permanent contact with Pb-free media did not reduce the basal amount of the metal in the blood of the toads (Arrieta et al. 2004).The reduction observed in this case-after a longer stage in clean media-may be attributed to depuration processes of the animals (Arrieta et al. 2004).
The information in relation to the impact of Pb on adult amphibians is scarce (Devilliers and Exbrayat 1992; Schuytema and Nebeker 1996;Linder and Grillitsch 2000;Rowe et al. 2003;Fink and Salibiµn 2005).A few authors have reported results that pointed out the peculiar tolerance of adult and larval amphibians to Pb (Kaplan et al. 1967;Birdsall et al. 1986;Vogiatzis and Lombourdis 1999;Rowe et al. 2001).The results presented in this article confirm our own previous finding, showing the tolerance of adult B. arenarum to Pb as was suggested by the acute 120 h-LD 50 (at 20°C), which was 89.2 mg Pb .100 g )1 body weight (Arrieta et al. 2000a).
It can be hypothesized that this tolerance may be related to the accumulation of the metal in the animal's target organs (liver, kidney, and spleen), and to the protective effect of the Pb binding proteins found mainly in kidney and erythrocytes (Fowler and Duval 1991;Conner and Fowler 1994;Quintanilla-Vega et al., 1995;Fowler and Squibb 1997;Bergdahl et al., 1998), The induction of those specific Pbprotective proteins in amphibians has not been reported yet.
In the cell counts of whole blood of Pb-injected toads (Table 2), the number of RBC showed a tendency to decrease, whereas a 100% increase in the total number of WBC was found (Table 1).In one of our previous studies, changes in the same sense were verified after 1 week of toads being injected with a single dose of 100 mg Pb .kg )1 , although the increase was not significant (Rosenberg et al. 2003).
a Results are expressed as g AE dl )1 (means € SD); in parentheses: number of samples; between brackets are relative values as percentage.
b vs c ; d vs e : p < 0.01; f vs h ; g vs h : p < 0.001 (Student's t test).Other comparisons were not statistically significant.
It is worth mentioning that these responses may be dose related; in another set of experiments carried out on the same toad species, in which a much lower dose of Pb was injected, the RBC and WBC counts did not change significatively (Perí et al. 1998a).It is also interesting to highlight that, contrary to what is found in humans, in adult B. arenarum, lymphocytes represented the highest percentage of leukocytes.
In relation to this, it was reported that the acute intoxication of larval amphibians also provoked anemia with destruction of mature RBC, as well as an increase in circulating immature cells; these results may be interpreted as an early compensatory stimulation of the erythropoiesis in the erythropoietic organs, thus preserving the oxygen transport capacity of blood (Dawson 1933;Barrett 1947).Particularly, in the case of Necturus sp., intoxication with Pb brought about a decrease in the number of circulating RBC with a subsequent presence and multiplication of erythroblastic forms in the blood flow (Dawson 1933).In addition, Rosenberg et al., (1998) demonstrated in B. arenarum that the osmotic fragility of the RBC resulted insignificantly reduced in Pb-injected toads.
A 27% decrease in the d-aminolevulinic acid dehydratase (d-ALAD) activity was found in B. arenarum, as a response to sublethal doses of Pb acetate.Furthermore, there was an almost nine-fold increase in the concentration of free erythrocyte protoporphyrins (Arrieta et al. 2004).
In relation to the alterations produced by Pb, a significant increase in the total leukocyte count was observed; among those alterations, there was an increase in the blast-like cells, lymphocytes, neutrophils, and monocytes, with the increase significant being only in the first cell type.Likewise, tendency to a decrease in the number of eosinophils was recorded.Absolute increases in the number of neutrophils and lymphocytes were responsible for the WBC count increase at the 6week period.This could be due to an induced proliferation, as a result of the metal's toxicity, of pluripotential hematopoietic cells that is a consequence of a depletion of circulating differential cells, as was suggested in studies by McMurry et al. (1995) regarding Pb intoxication on field rats.
The effects of Pb on the hematologic parameters of B. arenarum had already been studied in our laboratory (Peri et al. 1998b).After 3 weeks of having injected the animals with a 10 mg Pb .kg )l dose, the number of reticulocytes was found to increase greatly.The change found in the count of this element was suggested as being an early biomarker of intoxication due to Pb.
The tolerance to Pb shown by amphibians, in contrast with their sensitivity to Hg and Cd, can be interpreted as being a consequence of the specific conformational characteristics of critical enzymes, particularly those associated with the number of functionally -SH present in their structure, Those having a single -SH group generally require much higher metal concentrations to be inhibited (Valle and Ulmer 1972;Eisinger 1978).Because of its much greater mass/charge ratio, Pb may have greater affinity for specific sites and consequently displace, for instance, Zn in the d-ALAD, causing inhibition of the enzymatic activity.It has been proposed that d-ALAD might be responsible for a remarkable proportion of the Pb binding in RBC (Bergdahl et al. 1998).Campana et al., (2003) have recently reported that the injection of a single dose of Pb to a toadfish (Halobatrachus didactylus) increased the hepatic and renal metallothionein content, but the responses were not produced immediately, apparently depending on the time the metal takes to accumulate in the tissues.Results were interpreted as evidence of a lower sulfhydryl binding affinity to Pb compared with other heavy metals, such as Cd.
The results of the quantification and fractionation of the serum proteins of control toads was coincident with those reported by Bertini and Cei (1960), and were comparable to those informed in other amphibian species (Chalumeau-Le Foulgoc and Gallien 1967;Petrakis and Brown 1970).
When comparing both control and injected toads at the beginning and at the end of the experiment, a tendency to higher total proteins levels in a comparable dimension for both groups was found.This could be attributed to the protein input through the diet during each experimental period.In contrast, a significant decrease in total serum protein for both treatments at the end of the experiment was observed.If this decrease produces an alteration of oncotic pressure, it was not expressed as edema in Pb-injected toads, because we did not observe it.
The metal also causes nephrotoxic effects, These effects may explain the decrease in the total protein and in the albumin fraction concentrations found in Pb-injected toads with respect to contemporary control animals as a secondary effect of the adverse impacts of the metal on two of the main target organs of Pb.In this respect, it is worth mentioning the studies carried out in rats that have shown that Pb is bound to a-2-lglobulin fraction, which is synthesized in the liver (Fowler and DuVal 1991) protecting the heme pathway d-ALAD from Pb inhibition.
Pb is known to produce oxidative stress in human and animal cells with participation of several reactive oxygen species, increasing the lipid peroxidation in different tissues.Several adverse impacts of Pb may be interpreted on the basis of those effects.It was suggested that the resistance to oxidative stress and membrane lipid peroxidation could explain differences in the susceptibility to Pb among species (Mateo and Hoffman 2001).It is well known that prolonged exposure to Pb may cause adverse impacts on the liver cells due to impairment of the function of the hepatocytes, secondary to oxidative stress processes (Williams and latropoulos 2002).
Pb is known to alter the immune response of humans as well as of other animal species.Furthermore, the significant increase of the heaviest fraction of the globulins (G3) might be interpreted as a compensatory immunostimulation effect of the metal at the assayed dose.In this respect, it is interesting to note that Pb provoked either stimulation or inhibition of leukocytes in mammals, depending on their concentrations (Razani-Boroujerdi et al. 1999;Mishra et al. 2003).
Finally, it is interesting to incorporate into the discussion two ecotoxicological aspects.First the impact of chronic exposure of amphibians to heavy metals is a factor that may contribute to the decline of their natural periurban populations due to their depressive immunotoxicant effects (Boyer and Grue 1995;Rollins-Smith 1998;Carey et al. 1999) and the resulting increase in their susceptibility to a family of infections and infective factors (Christin et al. 2003).We have shown that in a short acute exposure, 1 week after one single 100 mg Pb .kg )1 injection to adult B. arenarum, a significant decrease of the phagocytic and lytic activity was found (Rosenberg et al. 2000(Rosenberg et al. , 2003)).These results may be contro-versial if compared with the increase of G3 plasmatic protein fraction, but it must be considered that this G3 increase was observed in long-term experiments in which toads, injected with higher Pb doses, had a different response.Second, because amphibians have short home ranges, they may be useful sentinels for detection of environmental Pb pollution in restricted areas through programs for monitoring the status of specific biosensors.

Fig. 1 .
Fig. 1.Electrophoretic fractions of seric proteins of male adult Bufo arenarum.Serum proteins electrophoretic fractions from male adult B. arenarum lead-injected and controls.Lane A: Control animal at the beginning of the experiment.Lane B: Control animal at the end of the experiment.Lane C: Injected animal at the beginning of the experiment.Lane D: Injected animal at the end of the experiment.Lane E: Human control serum

Table 1 .
Lead concentration in the whole blood of control and lead-injected male toads Bufo arenarum a Data are expressed as mg .dl )1 , mean € SD.In parentheses.number of animals T i initial time, T f final time.

Table 2 .
Total red (RBC) and white (WBC) blood counts of control and lead-injected male toads Bufo arenarum a a Data expressed as number of cells .ml )1 , mean € SD.In parentheses; number of animals.b vs c p < 0.001 (Student's t test).Other comparisons were not statistically significant.

Table 3 .
Differential leukocyte counts in the blood of control and leads-injected male toads Bufo arenarum a a Data are absolute values and are expressed as means € SD (• 10 7 ml )1 ).In parentheses; percentage values respect to total white blood cells counts.b vs c p < 0.01 (Student's t test).Other comparisons were not statistically significant.

Table 4 .
Total proteins and electrophoretic fractions of serum proteins of male toads Bufo arenarum a