ORIGINAL PAPER

Interaction effects of lead on bioavailabilityand pharmacokinetics of arsenic in the rat

Violet Diacomanolis • Barry N. Noller •

Jack C. Ng

Received: 3 January 2013 / Accepted: 26 March 2013 / Published online: 1 June 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Arsenic (As) and lead (Pb) are common

contaminants found in mine waste materials. For an

evidence-based risk assessment, it is important to better

understand the potential interaction of mixed contam-

inants; and this interaction study was investigated in an

in vivo rat model. Following co-administration of a

fixed dose of AsV as in sodium arsenate and different

doses of Pb as lead acetate to Sprague–Dawley rats,

blood arsenic concentration and bioavailability

decreased. A decrease in As blood concentration when

lead was co-administered was observed with increasing

lead doses. Pharmacokinetic parameters for As in the

blood showed faster absorption and elimination of this

metalloid in the presence of Pb. The elimination half-

life of As decreased from 67 days in As solo group to

27–30 with doses of Pb. Bioavailability of As was also

decreased by 30–43 % in the presence of Pb. Decreased

urinary excretion of Pb and tissue accumulation were

also observed. It indicates lower absorption of As when

co-administered with Pb. A probable explanation for

these findings is that As co-administration with Pb

could have resulted in the formation of less soluble lead

arsenate. However, such an interaction between As and

Pb could only explain about one-third of the variation

when real mine waste materials containing both of these

elements were administered to rats. This suggests that

other effects from physical and chemical parameters

could contribute to the bioavailability of arsenic in

complex real environmental samples.

Keywords Arsenic � Lead � Mine waste �Bioavailability � Mixed contaminants �Pharmacokinetic parameters � Interaction effect

Introduction

Contaminated sites often contain a mixture of metals/

metalloids and the interaction between them can affect

their bioavailability and toxic effect. Bioavailability of

metals/metalloids can be highly variable in popula-

tions because it is influenced by a variety of factors

including the chemical form of the metal/metalloid

(Andrews 2000), environmental matrix in which the

Electronic supplementary material The online version ofthis article (doi:10.1007/s10653-013-9527-x) containssupplementary material, which is available to authorized users.

V. Diacomanolis � J. C. Ng (&)

National Research Centre for Environmental Toxicity,

The University of Queensland, 39 Kessels Rd, Coopers

Plains, Brisbane, QLD 4108, Australia

e-mail: j.ng@uq.edu.au

V. Diacomanolis

e-mail: v.diacomanolis@uq.edu.au

B. N. Noller

Centre for Mine Land Rehabilitation, The University

of Queensland, St Lucia, Brisbane 4072, Australia

e-mail: b.noller@uq.edu.au

J. C. Ng

CRC for Contamination Assessment and Remediation

of the Environment, Mawson Lakes, Adelaide 5095,

Australia

123

Environ Geochem Health (2013) 35:757–766

DOI 10.1007/s10653-013-9527-x

ingested metal/metalloid is contained, gastrointestinal

tract contents, diet and nutritional status (Reeves and

Chaney 2002), genotype (Wright et al. 2004), age and

sex (Komarnicki 2000) and health status (Peakall and

Burger 2003; Ilback et al. 2004). Any significant

interactions between metals/metalloids that alter the

dose and hence the bioavailability to the organism,

need to be included in the risk assessment. However,

most dose–response assessments of metal/metalloid

mixtures have been based on the analysis of elemental

concentrations of individual metals/metalloids. The

risk for each is then added to give an overall risk. This

concept is termed additivity though simple addition of

the concentration and/or effects of single metal and

metalloid exposures may not accurately predict the

outcome of exposure to metals and metalloids mixtures

(Preston et al. 2000). Interactions between metals and

metalloids could include additive, synergistic, poten-

tiation or antagonistic effects.

Lead (Pb) and arsenic (As) are harmful metal and

metalloid to the environment, animals and humans. Of

these two elements, perhaps As has drawn the most

attention in that inorganic As is a proven human

carcinogen and well known for causing massive chronic

poisonings globally in recent history (Ng et al. 1999,

2003; Tsai et al. 2003; Milton et al. 2005; Ng 2005; Kapaj

et al. 2006; Ahsan et al. 2009; Argos et al. 2010). The

main source for As poisoning is of natural geological

source particularly from contaminated groundwater and

As-contaminated coal (Ng et al. 2003; Ng 2005). The

main sources for Pb toxicity include leaded petrol, paint

and battery and emission from smelting activity. Lead

and As are often found as co-contaminants in geogenic

materials (e.g. from mine and certain industrial sites).

The understanding of their respective individual in vivo

toxicity is well advanced. However, the interaction of

these two elements within a biological system and their

resulting potential for harm is less well studied. This

study aimed to examine the response of As in terms of its

bioavailability and other pharmacokinetic parameters in

the rat with and without co-administration of Pb.

Materials and methods

Reagents and consumables

All the chemicals were of analytical-reagent grade.

Sodium arsenic and lead acetate used for the rat dosing

were purchased from BDH Chemicals, Poole, Eng-

land, and Ajax Chemicals Ltd, Sydney, Australia,

respectively. All water used for sample preparation,

dilutions and dosing solution preparation was deion-

ised water (18 MX cm type I water, Milli-Q water

purification system, Millipore�, Billerica, MA, USA).

Nitric acid was reagent grade 69 % HNO3 (Analar�

Ajax Chemicals Ltd, Sydney, Australia). Dilution of

calibration standards, mine waste, urine and tissue

samples was made with 2 % HNO3 made from

concentrated HNO3 and deionised water. Dilution of

blood calibration standards and blood samples was

made with deionised water. All the glassware was

soaked in 10 % HNO3 for at least 24 h and then rinsed

thoroughly with deionised water and air-dried before

use.

Mine waste samples

Mine waste materials were collected from different

locations at two different mine sites in North Australia.

To have a representative sample of each area, 5–10

scoops (*200–250 g/scoop) of each surface soil sample

(0–10 cm depth) were collected in plastic bags and

composited (minimum 1 kg weight unless otherwise

stated). All mine waste materials were dried in a vacuum

oven for 10–12 h at 50 �C and sieved to \2 mm prior to

being ground in a zirconia TEMA swing mill with a

tungsten carbide mill for approximately 2.5 min to

reduce the particle size (PS) to below 250 lm; it is

generally accepted that smaller PS (i.e. 100–250 lm) is

more likely to adhere to children’s hands and be ingested

(Duggan et al. 1985). The ground mine waste samples

were digested in aqua regia, and digest solutions were

analysed for arsenic and lead concentrations by Induc-

tively Coupled Plasma-Mass Spectrometer (ICP-MS)

(Agilent 7500 CS, Tokyo, Japan).

Animal experiments

Animal experimentations were conducted under the

conditions approved by the Queensland Health Forensic

and Scientific Services Animal Ethics Committee (AEC

No. 07P05). Sprague–Dawley rats of 7–8 weeks of age

and approximately 200 g body weights were divided

into groups of at least 5 animals per treatment and were

quarantined for 5 days for acclimatisation prior to

experiments. Rats were fasted over night prior to dosing

to afford the most absorption of metals and metalloids

758 Environ Geochem Health (2013) 35:757–766

123

from their gastrointestinal tract. Rats were weighted,

marked and sampled for blood to give baseline samples

of blood via the tail vein before dosing.

Dose–response experiments

The appropriate dosage set for the animal studies was

selected based on doses below the LD50 in order to

avoid acute toxicity or mortality. Groups of fasted rats

were given As at 0.5 mg/kg (i.v.), 0.5, 5 or 15 mg/kg

(p.o.) as sodium arsenate in solution. Negative control

animals were given an equivalent volume of deionised

water. The dosing regime is shown in Table S-1

(online complementary information file).

Interaction experiments

For the interaction study of As and Pb, the dosing

regime is shown in Table S-1. Rats were given As at

2.5 mg/kg b.w. (p.o.) as sodium arsenate followed by

Pb at 0.5, 10 or 20 mg/kg b.w. (p.o.) as lead acetate

solution. Oral route was selected for the interaction

study as it reflects realistic scenario for arsenic and

lead transfer from mine sites in the environment. AsV

was selected for interaction experiments as it is the

dominant species found in oxidised environment.

Post-dosing rats were kept in individual metabolic

cages for 24 h pooled urine samples over the next

days. Periodic blood samples were also obtained.

Urinary and blood total elemental concentrations were

measured by ICP-MS. Additional detailed procedures

for rat dosing, rat sampling (blood, urine and tissue)

and rat sample preparation (blood, urine and tissue)

are given in Online Resources-Materials & Methods.

Analysis by inductive coupled plasma-mass

spectrometry (ICP-MS)

Arsenic and lead concentrations were measured by

ICP-MS (Agilent 7500 CS, Agilent Technologies,

Tokyo-Japan) together with certified reference mate-

rials (CRMs) for quality control of blood (Seronorm,

Level 2, Sero As, Sero, Norway), urine (Lyphocheck,

level 1, Bio-Rad, California, USA) and tissue samples

(SRM 1577b, freeze-dried bovine liver, Graham

B. Jackson, Melbourne, Australia). Calibration stan-

dards for ICP-MS were made up from Agilent multi-

element calibration standard 2A (Agilent Technologies,

Tokyo-Japan) (Bruce 2004; Huston 2005; Teijon et al.

2000). ICP-MS calibration solutions were diluted to the

required concentration of 2 % HNO3 for urine and

tissue samples and deionised water for blood samples.

The limit of detection within the 95 % CI was

0.32–0.88 ng/mL for arsenic and 0.61–1.12 for lead.

Quality control

To maintain operational quality control procedures,

internal standard (5 % HCl and 11 % HNO3 acid

containing 500 ng/mL Li, Sc, Ge, Y, In, Tb, Bi, Rh and

Au (20 ng/mL), supplied by Agilent for ICP-MS) was

used to check sample uptake and instrument drift

(Agilent Technologies 2003). Internal standard ele-

ments were also used as tools to overcome matrix

effects (Agilent Technologies 2003). Spike samples

also were used to check for recovery of As and Pb and to

use the recovery range for matrix interference correc-

tion (1 spike/20 urine/tissue samples and 2 spikes/48

blood samples). The recovery of As and Pb from

certified reference materials for blood, urine and tissue

samples was used to correct for recovery and to show

absence of matrix effect.

Data analysis

Blood pharmacokinetic parameters were calculated

with PK Solver, a freely available menu-driven add-in

program for Microsoft excel written in Visual basic for

applications (VBA), for pharmacokinetic and pharma-

codynamic (PK/PD) data analysis using blood elemen-

tal concentration–time data (Zhang et al. 2010).

GraphPad Prism (Version 5, GraphPad Inc., San

Diego, USA) was used to calculate the area under the

curve (AUC) of urine concentration over the 10 days

of experiment. This software was also used for the

linear and regression analysis for the blood, urine,

tissue and mine waste samples using 95 % CI.

Arsenic bioavailability was assessed using phar-

macokinetic analysis encompassing AUC blood

arsenic concentration time curve following back-

ground correction and dose normalisation of oral and

intravenously dosed animals. The dose–response

curve then was developed for As concentration in

blood, urine and tissues and the associated equations

used to calculate bioavailability and pharmacokinetic

parameters with and without co-administration of Pb.

SPSS for windows (version 17.0) was used for

general statistical analysis of results. Levene’s test was

Environ Geochem Health (2013) 35:757–766 759

123

used to test for equity of variances between groups. If

variances were not equal, a log normal transformation

was used to equalise variance and Tukey’s test was run

for such data. If variance was still unequal, a Dunnets

T3 test was used for post hoc analysis. Where group

sizes were unequal, the Bonferroni post hoc test was

used to compare the means of groups.

Results

Mine waste samples

Table S-2 (online complementary information file)

gives the results for the mine waste samples used in

this study. The arsenic concentrations range from

7–3,130 mg/kg, while lead is 118–866,000 mg/kg.

Thus, the mine waste samples cover a wide range of

possible concentrations that are likely to be found.

Arsenic dose–response relationship

To study arsenic kinetics in the blood of rats following

oral administration of three different doses of sodium

arsenate, blood arsenic pharmacokinetic parameters

were calculated to describe the distribution and elimi-

nation of As in rats. Figure 1a shows As concentration–

time curves for different doses of sodium arsenate.

Pharmacokinetic parameters were also calculated for

AsV intravenously dosed groups to use as the reference

for bioavailability calculation and for comparison with

the corresponding orally dosed groups. Based on Mann

et al.’s (Mann et al. 1996) PBPK model for arsenic

exposure in rabbits and hamster and also Gentry et al.’s

(Gentry et al. 2004) extrapolation of the Mann et al.

model for mice, arsenic absorption, distribution and

excretion in blood from both oral and intravenous routes

follow first-order kinetics. For the oral route, the ‘‘extra

vascular one-compartment model’’ was selected, and for

intravenous, the ‘‘i.v. bolus one-compartment model’’

was selected, to calculate arsenic pharmacokinetic

parameters from blood as those models had smaller

Akaike’s information criterion (AIC) compared to the

two- or non-compartmental model (Yamaoka et al.

1978; Ludden et al. 1994). The pharmacokinetic results

for the intravenous dosed groups are shown in Table S-3

(online complementary information file), for oral dosed

sodium arsenate in Table S-4 (online complementary

information file). A summary of As pharmacokinetic

data with and without the co-administration of Pb is

shown in Table 1.

Administering rats with different doses of arsenic

showed that the blood As concentrations were not

linear. It increased at lower doses following a linear

relationship and reached saturation at higher doses.

Nonlinear regression was used to derive the ED50

and also to determine maximum concentration in

blood. The ED50 for arsenic is the dose that creates the

half-maximum concentration of arsenic in blood. The

ED50 = 2.5 mg/kg b.w. shown by this model for AsV

dose–blood concentration as the effective dose needed

for AsV interaction experiments with lead. This dose

creates the half-maximum concentration of arsenic in

blood and is safe to prevent any possible adverse

effects due to interaction with lead. Figure 2a shows

the nonlinear regression modelling for rats’ blood

arsenic maximum concentration in response to

Fig. 1 Blood arsenic concentration–time curve for AsV as

sodium arsenate orally and 0.5 mg/kg intravenously adminis-

tered to rats (a). Blood arsenic concentration–time curve in

response to different doses of lead orally administered to rats

(b). Negative control group dosed with an equivalent volume of

deionised water and fed on normal rat feed (Mean ± SE,

n = 4–5)

760 Environ Geochem Health (2013) 35:757–766

123

different doses of sodium arsenate administered

orally. The nonlinear equation then was used to

calculate arsenic blood concentration for any given

dose. Other pharmacokinetic parameters were also

calculated using similar modelling (Table S-5, on-line

complementary information file).

Absolute bioavailability (ABA) of As was calcu-

lated using AUC for arsenic from the orally compared

to the intravenously dosed groups. Arsenic %ABA

decreased as the dose increased in both urine and

blood (Fig. 2b).

The decreasing trend of ABA against the increases

in doses of arsenic, however, was more sensitive in

blood compared to the urine. Both blood and urine

gave similar ABA results when the doses were

relatively low at about 0.5 mg/kg b.w.

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Fig. 2 Arsenic dose–response curve for rat blood maximum

arsenic concentration against different doses of AsV orally

administered to groups of rats (n = 4–5). *Statistically signif-

icant at p \ 0.01. Cmax and D are represented for maximum

concentration in blood and dose, respectively (a). Dose–

response in terms of absolute bioavailability (ABA) of arsenic

calculated from area under the curve of the blood and urine,

respectively, with the goodness of fit indicated by the r2 values

(b)

Environ Geochem Health (2013) 35:757–766 761

123

Arsenic interaction with lead

Following the co-administration of a fixed dose of

sodium arsenate (2.5 mg As/kg b.w.) and three

different doses of lead, the arsenic blood concentra-

tion–time curve was plotted for each As?Pb, dosed

group to show the effects of Pb (Fig. 1b). Blood

arsenic pharmacokinetic parameters were also calcu-

lated to describe how different doses of Pb affect

absorption, distribution and elimination of As in the

rat blood (Table S-5, online complementary informa-

tion file). The key pharmacokinetic values are sum-

marised in Table 1.

Co-administration of Pb and As increased the As

absorption (ka) and elimination rate (kio) and decreased

the half-lives of absorption (t�ka) and elimination (t�kio)

(p \ 0.01) which was significant at 20 mg/kg Pb co-

administered with 2.5 mg/kg As compared to 2.5 mg/kg

As solo. However, 20 mg/kg Pb co-administered with

2.5 mg/kg As had significantly higher absorption rate

(p \ 0.01) compared to other Pb?As and As solo

groups. Volume of distribution and clearance of As were

significantly higher in As?Pb dosed groups compared to

the As solo group (p \ 0.05). Maximum concentration

(Cmax), area under the blood concentration–time curve

(AUC) and bioavailability of arsenic from blood data

were significantly higher in the As solo group compared

to As?Pb dosed groups. This decreasing pattern was also

observed within the As?Pb dosed groups with increas-

ing lead dose (p \ 0.01).

Arsenic urinary excretion was also decreased in the

presence of Pb with increasing trend proportional to

increasing lead dose, compared to the As solo group

shown by significantly decreased concentration, per-

centage of dose and bioavailability of As in urine of

rats (p \ 0.05) (see also summary in Table 2).

Arsenic concentrations in the liver, kidney and

spleen of rats dosed with fixed dose of arsenic and

different doses of Pb showed decreasing trend with

increasing Pb doses showing significant differences

for the kidney and spleen (Fig. 3). The data suggested

that co-exposure of As and Pb had resulted in less As

was available for absorption.

Effect of environmental lead on bioavailability

and pharmacokinetic parameters of arsenic

Arsenic bioavailability in response to different doses of

Pb co-administered with fix dose of arsenic was plotted

for both blood and urine. Arsenic bioavailability of

mine waste samples was then calculated based on the

equations from As?Pb interaction to determine the

lead effects on bioavailability of arsenic from mine

waste samples. Bioavailability of arsenic from the rats

dosed with mine waste samples was then compared

against arsenic bioavailability values calculated from

arsenic dose–response equation in both blood and

urine. The entire mine wastes show significantly higher

Table 2 Arsenic urinary

excretions in rats dosed with

2.5 mg/kg arsenic solo or in

response to different doses of

lead given orally (Mean ± SE,

n = 4–5)

Dosed groups

(mg/kg)

As excretion As %Dose As %ABA

ng/24 h urine

As/2.5 1,01,000 ± 6,160 22.61 ± 1.44 65.63 ± 3.85

As?Pb/(2.5 ? 0.5) 81,500 ± 9,414 16.29 ± 1.88 39.74 ± 6.79

As?Pb/(2.5 ? 10) 37,100 ± 2,768 7.62 ± 0.55 13.47 ± 1.42

As?Pb/(2.5 ? 20) 26,500 ± 1,402 5.30 ± 0.28 9.05 ± 1.41

Fig. 3 Arsenic concentrations in the liver, kidney and spleen of

rats dosed with arsenic alone (2.5 mg/kg b.w.) or in combination

of lead at 0.5, 10 or 20 mg/kg b.w. Symbols * and ** indicate

statistical differences (p \ 0.05) between the control and

treatment groups

762 Environ Geochem Health (2013) 35:757–766

123

predicted bioavailability values compared to those

from the animal experiments. Regression analysis for

the predicted and experimental bioavailability values

indicated that Pb interaction effect explained only 25

and 35 % of the variations of As bioavailability using

blood and urine, respectively (Fig. 4).

Discussion

Arsenic was rapidly absorbed into the rat blood with a

peak concentration at 10-70 h, which did not decline

considerably over the experimental time (10 days).

This was shown by the concentration versus time

curve for rats dosed with sodium arsenate, orally and

intravenously and also in rats dosed with mine waste

samples contained arsenic. This phenomenon is due to

the relatively long half-life of As in rat blood, which is

about 60 days, and because As has an affinity to red

blood cells and binds to haemoglobin proteins (Suzuki

et al. 2004; Naranmandura et al. 2007, 2010).

Blood pharmacokinetic parameters determined by

different doses of As in rats (Table S-4, online

complementary information file) all supported the

physiologic characteristics of As in rat blood, includ-

ing fast absorption shown by ka and t�ka; relatively

persistent in blood as indicated by kio and t�kio; and

strong protein binding (Vd) and slow clearance from

blood (Cl). The higher absorption than elimination

constant (ka, kio) supported the fast absorption and

delayed elimination of arsenic in the rat blood.

Decreasing trend of absorption (ka) and increasing

elimination constant (kio) of arsenic proportional to

increasing doses showed that the As absorption and

elimination from blood were dose-dependent. This

observation is in agreement with Gonzalez et al.

(1995) who found decreased intestinal absorbed

arsenate with increasing doses after oral dosing of

3–240 mg As/kg b.w. No significant difference in

absorption and elimination of arsenic at higher doses

indicates a possible saturation point at higher doses of

AsV. It has been shown that there is a direct

relationship, although not proportional, between the

received dose and absorbed amount of arsenic as

intestinal absorption of arsenic reached a saturable

transport process (Gonzalez et al. 1995).

The bioavailability estimate of 0.5 mg/kg soluble

AsV orally dosed to rats (average 73.63 % as calcu-

lated from the blood) (Table S-4, online complemen-

tary information file) was in agreement with the

reported range of 70–98 % absorption coefficient of

soluble arsenic salts in mammals, including humans

(Owen 1990). This result is also supported by Rees

et al. (2009) who found nearly all the orally admin-

istered AsV doses to swine entered systemic circula-

tion of the animals with bioavailability values of

92.5 ± 22.3 %. Bioavailability as calculated from the

blood, however, decreased in higher doses (down to

10 %) and supported by decreased absorption constant

(ka). The decreasing ABA from lower to higher doses

supports a nonlinear dose-dependent absorption trend

of As from gastrointestinal tract into the blood; which

is in agreement with Gonzalez et al. (1995) who found

decreased intestinal absorbed arsenate with increasing

doses after oral dosing of 3–240 mg As/kg. Nonlinear

regression analysis confirms a curvilinear arsenic

dose-concentration response in blood with a high

goodness of fit (r2 = 0.91) (Fig. 2a). The same

nonlinear dose–response relation was found between

all other pharmacokinetic parameters in blood and

arsenic doses, including bioavailability. Nonlinear

dose-concentration response is an important point to

consider in arsenic risk assessment study that the

higher dose leads to lower bioavailability. A way to

avoid this problem is to select the dose appropriately

for both intravenous and oral routes of administration.

Fig. 4 Correlation between

predicted (based on As?Pb)

and experimental ABA of

arsenic from rat blood

(a) and urine (b). Line of

best fit (–) and 95 % CI (—),

*statistically significant at

p \ 0.05

Environ Geochem Health (2013) 35:757–766 763

123

Comparison of blood pharmacokinetic parameters

of the groups of rats dosed with arsenic and different

doses of lead showed increased absorption and

elimination of arsenic into the blood by increased

absorption (ka) and elimination rate (kio) and clearance

(Cl) from the blood, decreased half-life of absorption

(t�ka) and elimination (t�kio) and maximum residence

time (MRT) which resulted in decreased maximum

blood concentration of As (Cmax) and the subsequent

AUC in the presence of Pb. Decreased elimination

half-life of arsenic from 68 days in As solo group to

27–30 days in As?Pb dosed groups as shown in

Table 1 and Table S-5 (online complementary infor-

mation file) indicated faster elimination of arsenic in

the presence of Pb. The higher elimination rate and

clearance indicated efficient and rapid removal of As

from the body in the presence of lead.

Increased elimination rate and decreased elimina-

tion half-life were supported by consistently decreased

Tmax from more than 2 days in the As solo group to less

than 1 day in the presence of different doses of Pb in

As?Pb dosed groups. The higher elimination rate of

As is also supported by consistently increased arsenic

clearance from the blood of all As?Pb dosed groups

compared to As solo group. The higher elimination rate

and clearance indicated efficient and rapid removal of

arsenic from the body in the presence of lead.

Decreased concentration of As might be interpreted

as the mean that Pb co-administered with As decreased

As solubility in the gastro intestinal tract due to the

occurrence of chemical interaction between soluble

lead acetate and sodium arsenate, resulting in a less

soluble lead arsenate compound in the rat’s gut before

absorption into the blood. The form of insoluble

arsenical compound(s) as visible in the gut contents is

being confirmed by synchrotron X-ray spectroscopy

technique. In the lower alimentary tract with neutral

pH, where the absorption is taking place, lead arsenate

is much less soluble and hence will decrease its

absorption into the blood. It has been reported that

gastrointestinal absorption of low-solubility arsenic

compounds such as lead arsenate is much lower than

soluble inorganic arsenic (Yamauchi et al. 1986;

Marafante and Vahter 1987). Occurrence of interac-

tion probably decreased available free As by forming

insoluble Pb-As compounds in the gut of rats so

resulted in faster absorption of little available free As

into the blood followed by faster elimination of

absorbed-As from the blood.

Urinary excretion of As from rats dosed with As

solo or As?Pb showed decreasing trend with increas-

ing doses of Pb so the highest lead dose group (20 mg/kg)

had the lowest concentration and percentage of arsenic

dose in urine (Table 2). Decreased As urinary excre-

tion would normally be interpreted as meaning that Pb

lowers the absorption of As which is supported by

blood pharmacokinetic parameters. It is shown that

spontaneous formation of Pb-As compounds in the

gastrointestinal tract from sodium arsenate and lead

acetate could lower absorption of arsenic by 20–30 %

(Yamauchi et al. 1986) and, similarly, Pb and phos-

phorus (P) interact to decrease Pb bioavailability

(Scheckel and Ryan 2003). The behaviour of As and

Pb in an aqueous environment is similar to that of Pb

and phosphate and is due to the chemical similarity of

AsV and phosphate (Violante and Pigna 2002; Hetti-

arachchi et al. 2003; Scheckel and Ryan 2003).

Arsenic co-administered with Pb decreased arsenic

in the liver, kidney and spleen to the levels lower than

that of arsenic solo (Fig. 3). The higher concentration of

arsenic in the liver of rats dosed with 0.5 mg/kg Pb and

As, compare to As solo group, is probably due to the

very low dose of Pb in this group relative to considerable

contribution of dietary lead (40 %). Decreasing arsenic

stored in tissues of rats dosed with As?Pb compared to

arsenic solo group, apparently show decreased storage

of arsenic in tissues though the decreased percentage of

dose in tissues which is up to 3.6 % (from the highest

dose of Pb) (cumulative for liver, kidney and spleen) is

much less than decreased percentage of excretion in

urine which is 6.32–17.31 %. These results showed

that tissue accumulation of As with increasing Pb dose is

less than urinary excretion in all doses so As should

have been retained in body or have been excreted in

faeces.

Increased interaction of arsenic with the thiol

groups of cystein in proteins could lead to increased

arsenic retention in different tissues and organs

particularly those are rich in thiol groups such as

keratin protein in skin and hair. This also might be a

reason for decreased As in the liver at higher doses of

lead. The skin and hair could be a major source for As

accumulation especially in furry animals like the rat.

Skin is shown to contribute in As accumulation with

less than 5 % in human (Johnson and Farmer 1991).

Pentavalent As has a high affinity to the skeleton in

some animal species probably because of its chemical

similarity to phosphate (Vahter et al. 1983).

764 Environ Geochem Health (2013) 35:757–766

123

Increased faecal excretion, however, cannot be

proven as it was not measured in this project though it

might be very possible as rats are shown to have

extensive biliary excretion of inorganic As though it is

not a major elimination route for As in rats.

Higher predicted bioavailability values of mine

waste samples compared to those actually obtained

from animal experiments overestimated the bioavail-

ability of Pb from mine waste samples were probably

due to effects from the presence of other elements or

soil physicochemical factors which had not been

accounted for. The regression analysis showed the

effect of Pb accounted for 25 and 35 % of the variation

on the bioavailability of As as calculated from the

AUC of the blood and urine, respectively (Fig. 4).

Some other factors might have contributory effects on

arsenic bioavailability from mine waste samples

including the presence of other elements, metals and

metalloids in mine waste samples, and other inorganic

compounds and organic matters. All these factors

could suppress metal absorption from the solid matrix

of mine waste (Riethmuller et al. 2001; Trenfield et al.

2011).

Conclusion

Bioavailability of As as calculated from the AUC of

blood was more sensitive to variation of doses

compared to those obtained from the urine. Therefore,

it is important to match the dosage of pure As salt

solution and that of the sample matrix when blood is

used for the determination of bioavailability in rats.

Co-administration of Pb reduced the bioavailability of

As as demonstrated by reduced concentrations in the

blood, urine and tissues. Arsenic accumulation in

tissues reduced with increases in Pb doses. Formation

of insoluble lead acetate in the guts of rats resulting

from the interaction effects of lead acetate and sodium

arsenate might be the reason for decreased absorption

and consequently concentration of As in the blood,

urine and tissues of rats.

Additivity of dose or effect assumption for

co-exposure to As and Pb could result in over

estimation of risk. However, the effect of Pb could

only account for about 30 % of the variation in arsenic

bioavailability. Small number of animals might have

also contributed to large variation of PK parameters.

Further studies are needed with larger number of

animals, and the effect of other factors affecting the PK

parameters should also be explored.

Acknowledgments The project was funded by an ARC-

Linkage grant (LP0214185) and an APAI scholarship to V.D.

Access to the animal research facility at the Queensland Health

Forensic and Scientific Services is acknowledged. Entox is a

partnership between Queensland Health and the University of

Queensland.

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