JOURNAL OF APPLIED TOXICOLOGY

J. Appl. Toxicol. 21, 269–274 (2001)

DOI:10.1002/jat.753

Exposure of Rana ridibunda to Lead

II. Impact of Lead on Various Parameters of
Liver Metabolism of the Frog Rana ridibunda
Angelos K. Vogiatzis and Nikolaos S. Loumbourdis*
Department of Zoology, University of Thessaloniki, GR 54006 Thessaloniki, Greece

Key words: frog; Pb; liver; glycogen; glucose; fat; protein; lactate.

There are no data at present on the impact of lead (Pb) on amphibian metabolism, although declines
of amphibian populations due to man-made changes in the environment have been recorded in recent
years. We studied the impact of Pb on the liver metabolism of the frog Rana ridibunda by measuring
the hepatic levels of glycogen, lactate, total fat, protein and glucose. Liver is one of the primary target
organs of Pb bioaccumulation. Frogs were exposed for 4, 10 and 30 days to 14 mg l−1 of Pb (in the form
of Pb(NO3 )2 dissolved in water) and compared with matched controls. The level of glycogen in the liver
increased until day 30. The increase of the metabolite was time-dependent because there was a positive
correlation (r = 0.994, P = 0.006) between glycogen concentration and the days of Pb exposure. Lactate
concentration declined continuously up to day 30. Liver fat content decreased from day 10 to day 30. Protein
concentration declined continuously until day 30. Glucose concentration increased up to day 30. Glycogen
concentration was correlated negatively with liver fat content (r = −0.474, P = 0.005), liver protein content
(r = −0.562, P = 0.0004) and lactate concentration (r = −0.472, P = 0.005), whereas it was correlated
positively with the Pb burden of the liver (r = 0.578, P = 0.0005). The frog appeared to face a metabolic
stress over the 30 days of Pb exposure, without being able to control it. We concluded that the increase of
liver glycogen concentration was due to gluconeogenesis via lactate and lipolysis. Further experimentation
on key gluconeogenic and lipolytic enzymes over the 30 days of Pb exposure would elucidate the mechanisms
that may lead to such phenomena. Copyright  2001 John Wiley & Sons, Ltd.

elimination is very slow and there is hardly any microbial

INTRODUCTION
Over the last century a great number of flora and fauna
species were extinguished or are under a constant threat
of extinction. Anthropogenic factors are the main causes
of this phenomenon, with environmental pollution holding
the sceptres. Nriagu1 stated that environmental pollution
from heavy metals “is spreading like a silent epidemic”,
a fact that is indisputable.
Lead (Pb) has probably the widest distribution in the
human environment.2 It has been widely used in ancient
times in the manufacturing of drain pipes, cookery pots,
weapons and machinery. In modern times, Pb environ-
mental pollution is caused mainly by the industry of
batteries, sewage sludge applications in agriculture, min-
ing and smelting activities and vehicle and industry
exhausts, with the latter being fundamental in atmospheric
pollution.2 Institution of the EU directive regarding the
removal of vehicles from countries of the EU using leaded
fuel after 1 January 2000 is an attempt to control such
pollution.
Plants take up Pb from the soil. In animals, it accumu-
lates through the food chain over many years because Pb
* Correspondence to: N. S. Loumbourdis, Department of Zoology, Uni-
versity of Thessaloniki, GR 54006 Thessaloniki, Greece. E-mail:
Loubourd@bio.auth.gr
Copyright  2001 John Wiley & Sons, Ltd.
degradation.2 In vertebrates, over long periods of Pb expo-
sure, the metal is mainly accumulated in bone, whereas
over short-term exposure it is accumulated mainly in soft
tissues (kidneys, liver and the gastrointestinal tract) as well
as in red blood cells.3
The effects of Pb in living organisms are numerous and
very important. Lead toxicity causes haematological, gas-
trointestinal, and neurological dysfunction in mammals.
Severe or prolonged exposure may also cause chronic
nephropathy, hypertension and reproductive impairment.
Lead inhibits enzymes, alters cellular calcium metabolism
and slows nerve conduction.4
In our first study regarding Pb, we examined Pb bioac-
cumulation in various tissues of the frog Rana ridibunda.5
This frog is widely distributed in Europe. It is very com-
mon in the Balkans with a wide distribution in Greece.6
The purpose of that study was to evaluate the effect of Pb
in this frog under controlled conditions. Adult frogs were
exposed for 4, 10 and 30 days at a constant concentra-
tion of 14 ppm Pb (mg l−1 ) in the form of Pb(NO3 )2 . We
studied the accumulation of Pb in the liver, kidneys, skin
and gastrointestinal tract (GI tract), as well as the impact
of Pb on hepatic δ-aminolevulinic dehydratase (δ-ALAD).
At the end of the 30 days of exposure all tissues accumu-
lated Pb, whereas the hepatic δ-ALAD decreased by 90%
compared with control values.5
Received 17 January 2000
Revised 1 March 2001
Accepted 1 March 2001
270 A. K. VOGIATZIS AND N. S. LOUMBOURDIS
In the present study we represent our results regarding
the liver metabolism of the exposed frogs of the first
study. We measured the hepatic levels of glycogen, lactate,
total fats, protein and glucose. This frog, owing to its
wide distribution, can well serve as a bioindicator of
environmental pollution, because it meets all the criteria
listed by Lower and Kendall,7 a fact that we have shown
already in previous studies.5,8 – 10
Many frog populations are declining and extinction
has occurred in a few populations, caused by man-
made changes in the environment, including heavy metal
pollution.11 Data about Pb and energy metabolism are
either scarce, especially related to anurans like Rana ridi-
bunda, or none. In the present study our main goal was to
examine the effects of Pb on the liver metabolism of Rana
ridibunda in order to enrich our knowledge on the impact
of this particular heavy metal on the energy metabolism
of the frog.
EXPERIMENTAL
Test organisms
Experiments were conducted during the active period
of the animals in the summer of 1996. Adult female
frogs, Rana ridibunda, were purchased from a local dealer
who collected them from unpolluted areas of Macedo-
nia, Northern Greece. To acclimatize, frogs were kept in
plastic boxes (35 × 23 × 23.5 cm) in dechlorinated tap-
water at a depth of 2–3 cm for 6–7 days prior to the
experiments. The water was changed every 3 days and
boxes were cleaned thoroughly. Frogs were fed larvae of
Tenebrio molitor, raised in our laboratory under controlled
conditions. A total of 68 frogs were used in this study.
Animals were divided into the experimental group (33
animals) and the control group (35 animals).
Exposure experiments
Each group was placed in a plastic tank (120 × 65 ×
60 cm) presoaked overnight in 10% HNO3 (analyti-
cal grade). The concentration of the diluted Pb in the
experimental group was 14 ppm. This concentration cor-
responded to 1/10 of the 96 h LC50 for Pb.5 The Pb(NO3 )2
was prepared as a stock solution in deionized water.
Animals of the control group were kept in clean water
throughout the experiment. Before the addition of Pb in
the experimental group, a sampling of animals of both
groups (experimental and control) was done, which cor-
responded to 0 days of exposure. At the end of the 4th,
10th and 30th day of Pb exposure, three animal samplings
in each group were made.
Tissue preparations
Animals were sacrificed by a sudden strike at the head.
They were weighed to the nearest milligram and body
length was measured to the nearest millimetre. Liver sam-
ples were dissected and weighed to the nearest milligram,
and the hepatosomatic index (HSI) of each animal was
estimated (HSI = liver weight/body weight × 100). Sam-
ples were chilled in liquid nitrogen and then kept in a
Copyright  2001 John Wiley & Sons, Ltd.
freezer (−25 ° C) until required for the experiments. Tis-
sues were handled with plastic forceps and kept in plastic
bijou boxes.
Biochemical procedures
Small liver samples were thawed rapidly and boiled
in 3 ml of 30% KOH for 30 min. Two 1-ml samples
from each liver were analysed for glycogen with the
anthrone reagent12 using saturated Na2 SO4 , which acted
as a co-precipitant to improve glycogen recovery13 against
multiple glucose standards. Glycogen concentration was
expressed as mg g−1 wet tissue.
The estimations of glucose and lactate concentrations
were performed by the following procedures: 150–250 mg
liver samples were homogenized with a pestle and mor-
tar on ice with the addition of 10% HClO4 (10 : 1, v/w).
The homogenate was centrifuged at 5000 rpm at 4 ° C
for 10 min; 1 ml of the supernatant was neutralized with
ca. 0.6 ml of 3 M KHCO3 and centrifuged at 5000 rpm
at 4 ° C for 10 min. In the supernatant we estimated the
concentration of lactate and glucose using the method of
Hohorst14 and the glucose GOD-POD colorimetric testing
kit (Sigma kit no. 510), respectively. Lactate and glu-
cose concentration were expressed as µm g−1 wet tissue.
Total fats were estimated by the Folch et al. method15
and expressed as a percentage of wet weight tissue. Liver
proteins were determined by the Lowry et al. method16
using the supernatant of homogenized and centrifuged tis-
sue with the addition of phosphate buffer (10 : 1, w/v) at
pH 7.2.
Statistical analyses
The normality of the studied parameters was checked
using the Kolmogorov–Smirnov test, and because they
all followed a normal distribution the statistical analyses
were based on parametric tests. Values of the studied
parameters in each group were compared by analysis
of variance (ANOVA) and post hoc comparisons were
based on Dunnett’s multiple comparison tests. For the
correlation between the studied parameters we applied
Pearson’s r correlation. The comparisons of the studied
parameters between groups were based on Student’s t-
test (two-way) for independent samples. Differences were
deemed statistically significant at P < 0.05. Statistical
analyses were carried out with SPSS 7.51 for Windows.
RESULTS
Animals exposed to Pb were active during the experiment
and we did not observe any deaths or changes in animal
behaviour during the 30 days of Pb exposure.
We have not found any statistically significant differ-
ences between the gross morphological characteristics of
the experimental vs. control animals (Table 1).
In control animals, all the biochemical parameters stud-
ied remained unchanged over the 30 days of the experi-
ment (Table 2). In contrast, we noticed several changes in
the experimental animals due to their exposure to Pb.
Glycogen concentration in the liver appeared to increase
with the days of Pb exposure (Table 2). This increase
J. Appl. Toxicol. 21, 269–274 (2001)
 EXPOSURE OF RANA RIDIBUNDA TO LEAD 271

Table 1—Mean values (±SEM) of gross morphological characteristics of adult female Rana
ridibunda exposed (experimental group) or not (control group) to 14 ppm of Pb for 0, 4, 10
and 30 days (n = number of animals)

0 days in Pb 4 days 10 days 30 days

Experimental group
n 8 8 8 9
Body length 256.75 ± 4.03 248.75 ± 4.24 251.75 ± 5.72 246.44 ± 5.84
HSIa 3.02 ± 0.49 2.37 ± 0.16 2.92 ± 0.25 2.84 ± 0.18
Control group
n 9 8 9 9
Body length 250.11 ± 3.22 254.78 ± 5.02 253.33 ± 3.82 250.25 ± 4.39
HSIa 3.58 ± 0.28 2.97 ± 0.25 3.16 ± 0.42 3.12 ± 0.17

a
HSI = hepatosomatic index.

Table 2—Mean values (±SEM) of glycogen (mg g−1 ), lactate (µm g−1 ), fat (% of liver weight),
glucose (µm g−1 ) and proteins (mg g−1 ) in the liver of Rana ridibunda exposed (experimental group)
or not (control group) to 14 ppm of Pb for 0, 4, 10 and 30 days

0 days 4 days 10 days 30 days

Experimental group
Glycogen 21.59 ± 5.32c 36.29 ± 6.52d 41.84 ± 9.82e 87.55 ± 7.83∗
Lactate 3.34 ± 0.49b,c 2.93 ± 0.36d 1.99 ± 0.36 1.78 ± 0.22∗
Fat 8.10 ± 0.87c 6.39 ± 0.85 7.19 ± 0.58e 4.47 ± 0.74∗
Glucose 3.65 ± 0.44b,c 5.15 ± 0.26∗ 5.78 ± 0.78∗ 5.22 ± 0.46∗
Proteins 112.67 ± 3.46a,b,c 89.94 ± 1.55∗ 93.51 ± 3.94∗ 85.52 ± 3.67∗
Control group
Glycogen 27.53 ± 3.94 30.35 ± 3.49 32.84 ± 3.28 27.39 ± 4.45
Lactate 3.26 ± 0.42 2.74 ± 0.45 2.52 ± 0.44 3.56 ± 0.59
Fat 9.36 ± 0.56 8.88 ± 1.03 7.76 ± 0.84 8.57 ± 1.21
Glucose 3.39 ± 0.24 3.12 ± 0.21 2.25 ± 0.17 2.48 ± 0.22
Proteins 118.05 ± 5.84 109.37 ± 3.41 111.21 ± 5.21 110.43 ± 5.74

a,b,c
Values at 0 days were significantly different statistically from values at 4, 10 and 30 days in Pb,
respectively.
d
Values at 4 days in Pb were significantly different statistically from values at 30 days in Pb.
e
Values at 10 days in Pb were significantly different statistically from values at 30 days in Pb.
∗
Values of experimental animals were significantly different statistically from values of control animals.

appeared to be time-dependent because we found a posi-
tive correlation between liver glycogen concentration and
the time of exposure (r = 0.994, P = 0.006). Over the
first 10 days of Pb exposure the increase of glycogen con-
centration in the liver of the exposed animals was slower
compared with the rate of increase at the 30th day of Pb
exposure. Therefore, we did not find any statistically sig-
nificant differences compared with the values at 0 days of
exposure or with the respective values of the control ani-
mals (Table 2). The increase became more apparent after
the 10th day of exposure. At the 30th day of Pb expo-
sure, liver glycogen content increased by almost 400%
compared with the value at 0 days of exposure (Table 2).
The liver glycogen concentration value at the 30th day
of Pb exposure was significantly higher statistically com-
pared with the value at the respective days in clean water
(Table 2). In the experimental animals, glycogen concen-
tration was correlated positively with the Pb burden of the
liver (r = 0.578, P = 0.0005).
Lactate concentration decreased with the exposure of
the animals to Pb (Table 2). The decline in the lactate
concentration became statistically significant after the 4th
day of Pb exposure (Table 2). At the 10th day of Pb expo-
sure the lactate concentration decreased by 40% compared
Copyright  2001 John Wiley & Sons, Ltd.
with the value at 0 days of exposure. At the 30th day
of Pb exposure, lactate concentration was at the level
of the 10th day and it was significantly lower statis-
tically compared with the value at the respective days
in clean water (Table 2). At the 30th day of Pb expo-
sure we found a negative correlation between glycogen
and lactate concentration (r = −0.706, P = 0.033). In the
experimental animals, lactate concentration was corre-
lated negatively with the Pb concentration of the liver
(r = −0.486, P = 0.004) and the glycogen concentration
(r = −0.472, P = 0.005).
The liver fat content declined with exposure of the ani-
mals to Pb (Table 2). The decline became statistically
significant after the 10th day of Pb exposure. At the
10th day the fat content decreased by 11% compared
with the value at 0 days of exposure, whereas at the
30th day it decreased by 46%. The decline of the liver
fat content at the 30th day was statistically significant
compared with the respective value of the animals kept
in clean water (Table 2). At the 30th day of Pb expo-
sure we found a negative correlation between the liver fat
content and the glucose concentration (r = −0.752, P =
0.019). In the experimental animals the liver fat con-
tent was correlated negatively with the liver Pb content
J. Appl. Toxicol. 21, 269–274 (2001)
272 A. K. VOGIATZIS AND N. S. LOUMBOURDIS
(r = −0.474, P = 0.005) and the glycogen concentration
(r = −0.474, P = 0.005).
Glucose concentration increased with the exposure of
animals to Pb. This increase was statistically significant
compared with the value at 0 days of exposure and became
apparent from the 4th day of Pb exposure (Table 2). The
value of the glucose concentration at the 4th, 10th and
30th days was significantly higher statistically compared
with the values at the respective days in clean water
(Table 2).
Protein concentration decreased and remained constant
from the 4th day of Pb exposure onwards (Table 2).
The protein concentration in the liver of the animals
at the 4th, 10th and 30th days of Pb exposure was
significantly lower statistically compared with the val-
ues at the respective days in the group of the animals
kept in clean water (Table 2). The liver protein con-
tent was correlated negatively with the concentration of
Pb (r = −0.562, P = 0.0004) and the glycogen concen-
tration (r = −0.562, P = 0.0004) in the animals of the
group exposed to Pb.
DISCUSSION
This study is the second part of an experiment on the
effects of Pb on Rana ridibunda. In the first part we
studied the Pb bioaccumulation in various tissues of the
animal and hepatic δ-aminolevulinic dehydratase activity.5
In the second part we studied the Pb effects on liver
metabolism of adult frogs.
In the present study, both groups (experimental and
control group) were homogenous because we did not
observe any statistically significant difference between the
gross morphological characteristics.
We have not found any differences in the studied para-
meters of liver metabolism in the control group, therefore
we can speculate that all changes in the experimental
group were due to Pb exposure.
Exposure to Pb resulted in Pb accumulation (µg g−1 Pb
dry weight) in the liver of frogs (0 days: 7.78 ± 5.23;
4 days: 29.58 ± 8.0; 10 days: 56.87 ± 6.27; 30 days:
75.25 ± 6.08).5 The correlations between heavy metal
concentration in the liver and the studied parameters
of liver metabolism substantiate our conclusions. The
increase of intracellular Pb in the liver is responsible for
the critical changes observed.
In the previous study we showed that the ventral skin
was the tissue with the highest Pb content after 30 days
of exposure to 14 ppm of Pb dissolved in the water that
the frogs were kept in (Pb in the form of Pb(NO3 )2 ). Lead
cations entered the water-permeable skin and then via the
blood circulation was distributed to the soft tissues of the
frog, such as the liver.5 Divalent heavy metal ions like Pb
mimic the action and pathways of entrance of non-toxic
essential divalent ions such as Ca into the cell. It has been
shown that Pb enters into the cell using the Ca-pump or
Ca ion channels of cell membranes, resulting in an efflux
of Ca ions.17 – 20
Tulasi et al.21 studied the exposure of freshwater fish
Anabas testudineus to several sublethal concentrations
of Pb (1.25, 2.5, 5, 10 and 20 mg l−1 , in the form of
Pb(NO3 )2 ) for a period of 30 days. They found significant
Copyright  2001 John Wiley & Sons, Ltd.
accumulation of Pb in the blood and tissues. Lead bioaccu-
mulated in that study showed organ-specific distribution,
with high levels in the blood followed by the kidney, gill,
liver and brain and comparatively lesser amounts in the
ovary and muscle tissues. Exposure of the freshwater fish
Anabas testudineus to a sublethal (5 ppm) concentration
of Pb(NO3 )2 for a period of 30 days during the prepara-
tory phase of its annual reproductive cycle reduced the
total lipids, phospholipids and cholesterol levels in the
liver and ovary tissues but the free fatty acid levels were
increased and lipase activity was elevated.
Hacker et al.22 studied the effect of a single i.v. injec-
tion of Pb(NO3 )2 in rats. Male Wistar rats were given
a single i.v. injection of Pb(NO3 )2 (10 µmol 100 ml−1 )
and were killed with matched controls 24 h, 48 h, 72 h
and 20 days after the treatment. Changes of liver car-
bohydrate metabolism were studied histochemically by
testing the following parameters: glycogen content and
activities of glycogen synthase, glycogen phosphorylase,
glucose-6-phosphatase, glucose-6-phosphate dehydroge-
nase, 6-phosphogluconate dehydrogenase and glyceral-
dehyde-3-phosphate dehydrogenase. Between 24 and 48 h
after Pb(NO3 )2 injection there was a nearly complete
loss of liver glycogen whereas the activities of glycogen
synthase and glycogen phosphorylase were diminished.
Seventy-two hours later the polysaccharide reappeared in
single hepatocytes and after 20 days the livers of the lead-
treated animals not only had replenished their glycogen
stores but contained even more glycogen than the matched
controls, whereas the enzymes responsible for glycogen
formation and degradation restored their activities at nor-
mal levels.
We could speculate with no doubt that in the frog Rana
ridibunda what takes place is gluconeogenesis, which
becomes statistically significant after 4 days of Pb expo-
sure. Glucose concentration values in the experimental
group are significantly higher statistically compared with
glucose values of matched controls, even from the first 4
days of Pb exposure (Table 2). The negative correlations
between glycogen and lactate and fat content and glucose
concentration in the experimental group support this idea.
Degradation of fat to fatty acids and glycerol may lead to
gluconeogenesis via glycerol. By the end of the experi-
ment the fat liver content decreased to 50% compared
with the values at 0 days of exposure (Table 2). Glucose
concentration exhibited a constant increase in the range
41–58% compared with the value at 0 days of exposure
in the experimental group (Table 2). Increase in gluconeo-
genesis most probably leads to glycogen synthesis because
it is well known than Pb inhibits the action of glucose-6-
phosphatase, the enzyme responsible for glucose dimina-
tion from the hepatocyte.23 – 25 An intracellular increased
concentration of glucose-6-phosphate results in inactiva-
tion of glycogen phosphorylase (inhibition of glycogen
degradation) and activation of glycogen synthase (glyco-
gen synthesis).26
Protein liver content decreased during exposure in the
experimental group (Table 2). The decline that we found
was in the range 17–24% compared with the value at 0
days of exposure. We also observed a negative correlation
between the protein and glycogen content of the liver,
as well as between the liver protein content and Pb
concentration in the liver of the animals in the experi-
mental group. Gluconeogenesis via protein degradation
is a procedure that can take place in the hepatocyte.
J. Appl. Toxicol. 21, 269–274 (2001)
 EXPOSURE OF RANA RIDIBUNDA TO LEAD
Nevertheless, degradation of proteins to form glucose is
considered the ultimate action of an organism in order
to provide energy. Another explanation for the decline
of protein liver content could be the proteinuria (loss of
proteins via malfunction of kidneys) that is observed in Pb
toxicosis.27,28 Even so, we cannot eliminate the possibility
of protein degradation due to glucose formation.
We do not know what the mechanism is that impels
the hepatocyte to gluconeogenesis. Data from international
references regarding heavy metals and metabolism, espe-
cially emphasizing the effects of Pb, are scarce if at all.
However, glucosuria may be the mechanism.28
In a previous paper we studied the effects of Cd on the
liver metabolism of Rana ridibunda.10 Frogs were exposed
for 4, 10 and 30 days in 200 mg l−1 of Cd (in the form
CdCl2 ) dissolved in water. The level of glycogen in the
liver increased up to day 10, with a slight decrease at day
30. Lactate concentration was constant until day 10 but
then increased at day 30. Liver fat content decreased from
day 10 to day 30. Protein concentration was elevated at
day 10 but decreased up to day 30. The frog appeared
to face a metabolic stress (as in the case of Pb) over
the first 10 days of Cd exposure. At the 30th day of
Cd exposure the liver metabolism appeared to return to
normal conditions, most probably due to activation of
the defensive mechanisms of the organism against Cd
toxicity, namely an increase in the liver metallothioneins
and glutathione concentration.
In the case of Pb we have a similar situation. Exposure
of adult frogs to Pb results in lipolysis and gluconeo-
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273
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experiment. We do not know what happens after this time.
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