Download Response of glucose metabolism enzymes in an acute porphyria

Toxicology 216 (2005) 49–58
Response of glucose metabolism enzymes in
an acute porphyria model
Role of reactive oxygen species
Sandra M. Lelli, Leonor C. San Martı́n de Viale, Marta B. Mazzetti ∗
Laboratorio de Disturbios Metabólicos por Xenobióticos, Salud Humana y Medio Ambiente (DIMXSA), Departamento de Quı́mica Biológica,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
Pab. II, 4to Piso, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina
Received 4 March 2005; received in revised form 11 July 2005; accepted 19 July 2005
Available online 24 August 2005
Abstract
Acute hepatic porphyrias are human metabolic diseases characterized by the accumulation of heme precursors, such as 5aminolevulinic acid (ALA). The administration of glucose can prevent the symptomatology of these diseases. The aim of this work
was to study the relationship between glucose metabolism disturbances and the development of experimental acute hepatic porphyria,
as well as the role of reactive oxygen species (ROS) through assays on hepatic key gluconeogenic and glycogenolytic enzymes;
phosphoenolpyruvate carboxykinase (PEPCK) and glycogen phosphorylase (GP), respectively. Female Wistar rats were treated with
three different doses of the porphyrinogenic drug 2-allyl-2-isopropylacetamide (AIA) and with a single dose of 3,5-diethoxycarbonyl1,4-dihydrocollidine (DDC). Thus, rats were divided into the following groups: group L (100 mg AIA + 50 mg DDC/kg body wt.);
group M (250 mg AIA + 50 mg DDC/kg body wt.) and group H (500 mg AIA + 50 mg DDC/kg body wt.). The control group (group
C) only received vehicles (saline solution and corn oil). Acute hepatic porphyria markers ALA-synthase (ALA-S) and ferrochelatase,
heme precursors ALA and porphobilinogen (PBG), and oxidative stress markers superoxide dismutase (SOD) and catalase (CAT)
were also measured in hepatic tissue. On the other hand, hepatic cytosolic protein carbonyl content, lipid peroxidation and urinary
chemiluminescence were determined as in vivo oxidative damage markers. All these parameters were studied in relation to the
different doses of AIA/DDC. Results showed that enzymes were affected in a drug–dose-dependent way. PEPCK activity decreased
about 30% in group H with respect to groups C and L, whereas GP activity decreased 53 and 38% in group H when compared to
groups C and L, respectively. On the other hand, cytosolic protein carbonyl content increased three-fold in group H with respect
to group C. A marked increase in urinary chemiluminescence and a definite increase in lipid peroxidation were also detected. The
activity of liver antioxidant enzyme SOD showed an induction of about 235% in group H when compared to group C, whereas
CAT activity diminished due to heme depletion caused by both drugs. Based on these results, we can speculate that the alterations
Abbreviations: AIA, 2-allyl-2-isopropylacetamide; ALA, 5-aminolevulinic acid; ALA-S, ALA-synthase; CAT, catalase; DDC, 3,5diethoxycarbonyl-1,4-dihydrocollidine; DNPH, 2,4-dinitrophenylhydrazine; GP, glycogen phosphorylase; HCB, hexachlorobenzene; H2 O2 , hydrogen peroxide; HO• , hydroxyl radical; LDH, lactic dehydrogenase; MDA, malondialdehyde; NADH, ␤-nicotinamide adenine dinucleotide; PBG,
porphobilinogen; PBG-D, porphobilinogen deaminase; PCT, porphyria cutanea tarda; PEPCK, phosphoenol pyruvate carboxykinase; ROS, reactive
oxygen species; O2 • , superoxide anion; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances
∗ Corresponding author. Tel./fax: +54 11 4576 3342.
E-mail addresses: [email protected], [email protected] (M.B. Mazzetti).
0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2005.07.016
50
S.M. Lelli et al. / Toxicology 216 (2005) 49–58
observed in glucose metabolism enzymes could be partly related to the damage caused by ROS on their enzymatic protein structures,
suggesting that they could be also linked to the beneficial role of glucose administration in acute hepatic porphyria cases.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Acute porphyria model; Glucose metabolism; Reactive oxygen species; Rat liver
1. Introduction
The hepatic heme biosynthetic pathway is closely regulated, and intermediates 5-aminolevulinic acid (ALA)
and porphobilinogen (PBG), as well as porphyrins, the
oxidized forms of porphyrinogen, are excreted only
in trace amounts. However, this regulation is altered
in acute hepatic porphyrias, and the activity of ALAsynthase (ALA-S), the rate limiting step of the pathway,
feedback-regulated by intracellular heme concentration,
is increased, causing the overproduction and subsequent
accumulation and excretion of porphyrins, their precursors, or both (Kappas et al., 1995). The induction
response of hepatic ALA-S elicited by certain chemicals
can be enhanced by concurrent heme depletion and heme
synthesis inhibition (Granick, 1966).
ALA has been associated with iron-mediated oxidative damage to biomolecules and cell structures (Rocha
et al., 2003). Moreover, this compound is susceptible
to metal catalyzed oxidation, producing reactive oxygen
species (ROS) such as superoxide anion (O2 •− ), hydrogen peroxide (H2 O2 ), hydroxyl radical (• OH), and ALA
enoyl-radical (• ALA) (Monteiro et al., 1989), which
act as pro-oxidants both in vivo and in vitro (Bechara,
1995; Demasi et al., 1996). ROS are able to oxidize
nucleic acids, proteins, lipids, or carbohydrates, inactivating key cellular functions (Halliwell and Gutteridge,
1999).
Through the years, the administration of glucose to
porphyric patients has been reported to produce significant clinical progress (Doss et al., 1985; Kappas et al.,
1995). The prevention of acute experimental porphyria
through high carbohydrate and/or protein intake is an
example of the effect of glucose on ALA-S, in which
carbohydrates prevent its induction (Granick, 1966).
Schoenfeld et al. (1985) observed that the impairment
of ALA-S induction by 2-allyl-2-isopropylacetamide
(AIA) occurred in the presence of glucose. Likewise,
Rose et al. (1961) found that high glucose intake
prevented the development of experimental porphyria
induced by AIA, whereas fat intake had no effects whatsoever.
On the other hand, Correia and Lunetta (1989)
reported that gluconeogenesis was altered in rats
with porphyria induced by phenobarbital and 4-
ethyldihydropyridine, which are compounds that jointly
produced significant heme depletion. They also caused
gluconeogenic blockage, which – according to these
authors – might be the underlying mechanism of the
“glucose effect” in hepatic porphyria.
AIA and 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC) produce significant heme depletion and increase
ALA-S activity leading to ALA accumulation. Whereas
DDC inhibits ferrochelatase, the heme synthesizing
enzyme, causing protoporphyrin IX accumulation, AIA
destroys cytochrome P-450 heme (Smith and De Matteis,
1980; Marks et al., 1988). Both effects significantly
decrease the regulatory heme pool. Heme exerts a negative feedback control on ALA-S activity, and in order to
meet the need for cytochrome P-450 synthesis, ALA-S
induction occurs as a consequence of this heme depletion
condition (Whiting and Granick, 1976).
Taking into account the reports already mentioned
on the effects of porphyrinogenic drugs on glucose
metabolism, and due to the fact that AIA/DDC triggers an experimental porphyria similar to acute human
porphyria, we considered it important to analyze the relation between the porphyria and the alterations caused by
the drugs in different glucose metabolism enzymes. The
aim of this study was to investigate the effect of oxidative damage induced by acute intoxication with AIA and
DDC on the glucose metabolism of rats and its relation
to the degree of porphyria developed.
2. Materials and methods
2.1. Materials
AIA was a gift from Roche Co. (Germany). DDC
was purchased from Aldrich Chemical Company Inc.
(Milwaukee, WI). ALA, 5 -adenosine monophosphate,
ascorbic acid, bovine serum albumin, deoxyguanosine 5 -diphosphate, epinephrine, glucose-1-phosphate,
glycogen type III from rabbit liver, LDH, MoNH4 ,
malic dehydrogenase, MDA, NADH, pyruvate, thiobarbituric acid, Dowex 1 and Dowex 50W were obtained
from Sigma Chemical Co. (St. Louis, MO). Guanidine
hydrochloride was obtained from Invitrogen (Carlsbad,
CA). All other chemicals were of analytical grade.
S.M. Lelli et al. / Toxicology 216 (2005) 49–58
2.2. Animals and treatment
Female Wistar rats (180–200 g) were purchased from
the National Committee of Atomic Energy (CONEAArgentina). They were maintained on food and water
ad libitum and housed under conditions of controlled
temperature (25 ◦ C) and light (12 h light–dark cycle,
light from 06:00 to 18:00 h). Rats were fasted 24 h (8 h
before treatment and 16 h after treatment) before sacrifice. Total urine was collected during 16 h after intoxication using metabolic cages. Animals were treated
in accordance with guidelines from the Animal Care
and Use Committee of the Argentine Association of
Specialists in Laboratory Animals (AADEALC), and
the Guide to the Care and Use of Experimental Animals issued by the Argentine Council on Animal
Care.
AIA was dissolved in saline solution (0.15%, w/v)
(De Matteis, 1970) and DDC was dissolved in 2.5 ml
corn oil (De Matteis et al., 1973). Both solutions
were prepared just before administration. Rats were
fasted 8 h before treatment with both drugs. AIA was
injected subcutaneously (sc) in three different doses and
DDC, intraperitoneally (ip) in a single dose. Thus, animals were divided into the following groups: group
L (100 mg AIA + 50 mg DDC/kg body wt.), group M
(250 mg AIA + 50 mg DDC/kg body wt.), and group H
(500 mg AIA + 50 mg DDC/kg body wt.). Control group
(group C) only received vehicles: saline solution, sc and
corn oil, ip. These AIA and DDC doses were previously
used by other research groups (De Matteis, 1970; De
Matteis et al., 1973) in order to obtain porphyria models
in rats.
2.3. Liver preparation and assays
Rats were decapitated 16 h after AIA/DDC administration. All assays were performed in rats with a total
fasted period of 24 h, since this is the optimum way to
measure PEPCK and GP activities, as demonstrated by
Mazzetti et al. (2004) and Taira et al. (2004). This fasting
period enhances the sensitivity of the assays, permitting
the standardization of results obtained respect to the control. On the other hand, since the effect of AIA depends
on the previous diet (Tschudy et al., 1964), the 8 h starvation period prior to intoxication guaranteed that the
effect of the drug was not altered by the diet. A fraction
of liver (1 g) was excised and immediately homogenized (1:3 w/v) in a solution containing 0.9% NaCl,
0.1 mM Tris–HCl pH 7.4, and 0.5 mM EDTA to determine ALA-S activity, and ALA and PBG hepatic content
(Marver et al., 1966). The rest of the organ, previously
51
perfused with ice-cold saline solution, was removed and
homogenized in a Potter-Elvehejm homogenizer using
different solutions. To determine PEPCK activity, liver
was homogenized (1:3 w/v) in 0.25 M sucrose at 0–4 ◦ C.
The homogenate was centrifuged for 1 h at 100,000 × g,
and the resulting supernatant was used for PEPCK determination. In the case of GP, livers were homogenized
in three volumes of 0.25 M sucrose and 1 mM EDTA.
The homogenate was centrifuged for 10 min at 2000 × g
and the resulting supernatant was used for enzymatic determination. All procedures were carried out
at 4 ◦ C.
To measure CAT and total SOD activities, liver
homogenates were prepared (1:10 w/v) in a solution containing 140 mM KCl and 25 mM potassium phosphate
buffer (pH 7.4), then they were centrifuged at 900 × g
for 15 min. The resulting supernatant, a suspension of
preserved organelles, was used for enzymatic determinations.
For hepatic ferrochelatase determination, homogenates were prepared with 0.154 M KCl (1:5 w/v).
2.3.1. 5-Aminolevulinic acid synthase
ALA-S hepatic activity was assayed in whole
homogenates by the method of Marver et al. (1966).
Incubation mixtures containing 0.1 M glycine, 0.01 M
EDTA, 0.08 M Tris–HCl buffer pH 7.2, and 0.5 ml of
homogenate, in a final volume of 2 ml, were incubated
at 37 ◦ C for 60 min. The product was determined spectrophotometrically at 553 nm as detailed in Mauzerall
and Granick (1956).
2.3.2. 5-Aminolevulinic acid and porphobilinogen
hepatic contents
To determine ALA and PBG hepatic content, portions
of whole liver homogenates (Marver et al., 1966) were
deproteinised with 0.6 M TCA. Supernatants (0.3–1 ml)
were adjusted to pH 4.5–6.0. Contents were essentially
determined by the method of Piper et al. (1973). Two separate columns were used; PBG was eluted from a Dowex
1 column with 1 M acetic acid, whereas ALA was eluted
from a Dowex 50W column with 1 M sodium acetate
after removing urea. ALA pyrrole and PBG were measured colorimetrically as described by Mauzerall and
Granick (1956).
2.3.3. Ferrochelatase activity
Ferrochelatase activity was measured in a mitochondrial fraction. Pellets were frozen at −20 ◦ C for 24–48 h.
Enzymatic activity was measured using protoporphyrin
IX as substrate, as described by Porra and Jones (1963).
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S.M. Lelli et al. / Toxicology 216 (2005) 49–58
2.3.4. Phosphoenolpyruvate carboxykinase activity
PEPCK was measured using deoxyguanosine 5 diphosphate as nucleotide substrate. A 50 ␮l aliquot
of 100,000 × g supernatant was used in each assay.
Oxaloacetate formed during the reverse enzyme reaction
was determined by reduction with malic dehydrogenase in the presence of NADH. Changes in absorbance
were measured spectrophotometrically at 340 nm. The
reaction was allowed to proceed for 2 min at room temperature (Petrescu et al., 1979).
2.3.5. Glycogen phosphorylase activity
GP activity was assayed according to Newman and
Armstrong (1978). Final reagent concentrations were
50 mM glucose-1-phosphate, 1% rabbit liver glycogen III, 0.15 M NaF, 1 mM 5 -AMP, 0.5 M Na2 SO4 ,
0.5 M citrate buffer pH 6.5. Activity was measured
as released inorganic phosphate spectrophotometrically
determined at 660 nm through the phosphomolybdate
complex formed using ascorbic acid as reducer (Ayes,
1966).
2.3.6. Protein carbonyl content
Carbonyl groups formed on cytosol proteins were
quantified adding 1 ml 10 mM DNPH in 2 M HCl to a
cytosol volume containing 2 mg protein (200 ␮l approximately). They were allowed to stand in the dark at room
temperature for 1 h, with vortex mixing every 10 min.
Samples were precipitated with TCA (20% final concentration) and centrifuged in a tabletop micro-centrifuge
for 5 min. Supernatants were discarded and protein pellets were washed twice with TCA and then three times
with 1 ml of ethanol/ethyl acetate (1:1) to remove free
DNPH. Protein samples were resuspended in 1 ml 6 M
guanidine hydrochloride, pH 2.3, at 37 ◦ C for 15 min
with vortex mixing. Carbonyl contents were determined
measuring absorbance at 390 nm with a molar absorption
coefficient = 22,000 M−1 cm−1 (Levine et al., 1994).
2.3.7. Lipid peroxidation
Lipid peroxidation in liver was determined measuring
TBARS production rate. The red MDA–thiobarbituric
acid complex produced was extracted with butanol
to avoid the interference of endogenous porphyrins. TBARS content was determined measuring
absorbance at 532 nm with a molar absorption coefficient = 156,000 M−1 cm−1 (Ohkawa et al., 1979).
2.3.8. Liver antioxidant defense system enzymes
CAT activity was measured recording the decrease in
H2 O2 absorbance at 240 nm (Chance et al., 1979). SOD
activity, on the other hand, was determined by the rate of
autoxidation of epinephrine to adenochrome (Misra and
Fridovich, 1972). In this assay, a unit is defined as the
amount of SOD necessary to inhibit 50% epinephrine
autoxidation.
2.4. Rat urinary chemiluminescence
Total urine was collected for 16 h after intoxication
using metabolic cages. It was homogenized and kept
frozen until evaluation. Then, 1 ml urine was centrifuged
at 300 × g for 10 min (Rı́os de Molina et al., 1998).
Chemiluminescence was quantified using a Beckman
LS-3150P liquid scintillation counter operated in the outof-coincidence mode.
2.5. Protein determination
Protein concentration was measured following Lowry
et al. (1951) using bovine serum albumin as standard.
Protein concentration in carbonyl content assay was
determined in guanidine-dissolved samples measuring
absorption at 280 nm. The amount of protein was calculated from a bovine serum albumin standard curve
(0.25–2 mg/ml) dissolved in guanidine hydrochloride
and read at 280 nm (Reznick and Packer, 1994).
2.6. Statistical analysis
Values expressed in figures and tables are the means
of three different experiments performed in duplicate,
and involving two animals each. They indicate mean values ± S.E.M. Data were submitted to one-way ANOVA.
When the overall F-statistic was significant, multiple comparisons among groups were performed using
Tukey–Kramer’s test. The level of significance used was
P < 0.05.
3. Results
3.1. Heme pathway biosynthesis and precursors
Changes in porphyrin biosynthesis were studied using
different AIA/DDC doses to evaluate their effects on
some parameters of the acute intoxication model. Thus,
the activities of ALA-S and ferrochelatase (key enzymes
of the porphyrin pathway), and the contents of ALA
and PBG in liver were analyzed (Table 1). AIA/DDC
administration enhanced ALA-S activity up to 300%
in group H (500 mg AIA + 50 mg DDC/kg body wt.), and
this enhancement statistically differed from groups
C (control), L (100 mg AIA + 50 mg DDC/kg body wt.),
and M (250 mg AIA + 50 mg DDC/kg body wt.). ALA
S.M. Lelli et al. / Toxicology 216 (2005) 49–58
53
Table 1
Dose–response of hepatic ALA-S and ferrochelatase activities, and ALA and PBG hepatic contents to AIA/DDC treatment
Control group C
AIA + DDC treated
Group L
ALA-synthase (nmol ALA/g h)
Ferrochelatase (nmol heme/mg h)
ALA hepatic content (nmol ALA/g)
PBG heptic content (nmol PBG/g)
25.5
1.37
7.5
0.32
±
±
±
±
2.7
0.23
0.7
0.02
33.90
0.62
9.30
2.43
Group M
±
± 0.19*
± 0.90*
± 0.03*
4.9*
80.4
0.69
26.5
12.7
±
± 0.21*
± 1.7#,*
± 1.1#,*
7.7#,*
Group H
85.1
0.79
28.2
16.4
±
±
±
±
6.7§,#,*
0.22*
1.9#,*
1.7§,#,*
Animals were injected subcutaneously (sc) with three different doses of AIA and intraperitoneally (ip) with a single dose of DDC, divided into
the following groups: group L (100 mg AIA + 50 mg DDC/kg body wt.), group M (250 mg AIA + 50 mg DDC/kg body wt.), and group H (500 mg
AIA + 50 mg DDC/kg body wt.). Control group (group C) only received vehicles: saline solution, sc and corn oil, ip. Each value represents the
mean ± S.E.M. of six animals. Activity is expressed as nmol ALA/g wet liver h. Hepatic contents are expressed as nmol/ g wet liver.
* P < 0.05 is significantly different from group C. Multiple comparisons among treatment groups were performed using Tukey–Kramer’s test.
# P < 0.05 is significantly different from group L. Multiple comparisons among treatment groups were performed using Tukey–Kramer’s test.
§ P < 0.05 is significantly different from group M. Multiple comparisons among treatment groups were performed using Tukey–Kramer’s test.
and PBG hepatic contents in group H increased 276 and
5025%, respectively, when compared to C, and were statistically different from groups C and L. In addition, PBG
content in group H was statistically different from group
M. On the other hand, ferrochelatase activity decreased
about 50% in all the treated groups with respect to the
control.
3.2. PEPCK and GP activities
As regards urinary chemiluminescence, values
showed a statistically significant increase; 127% in
group L ((116.8 ± 38) × 103 cpm) and 205% in group
M ((156.2 ± 60) × 103 cpm) with respect to the control
group ((51.7 ± 25) × 103 cpm); P < 0.05. On the other
hand, animals treated with the highest dose (group H)
fell deeply asleep due to the treatment and, therefore,
the urinary excretion volume was not enough for chemiluminescence determination to be performed.
Figs. 1 and 2 show PEPCK and GP activities, respectively, measured in the liver of animals treated with
different AIA/DDC doses, as well as in controls. After
16 h of treatment, PEPCK suffered 30 and 24% inhibition in groups H and M, respectively, when compared
to controls and are statistically different from groups C
and L. GP activity also showed statistically significant
decreases of about 53 and 38% in group H with respect
to group C and group L, respectively. In group M, GP
suffered 36% inhibition when compared to group C.
3.3. Oxidative stress parameters
ROS are responsible for protein carbonylation and
membrane peroxidation damage. After the 16 h treatment, cytosolic carbonylated proteins suffered statistically significant 2.3- and 3.0-fold increases in groups M
and H, respectively, when compared to the control, and
were statistically higher than group L (Fig. 3).
Lipid peroxidation measured as TBARS exhibited a
statistically significant increase in group L (9%), group
M (24%) and group H (52%) with respect to the control
group, showing a drug–dose-dependent behavior in the
multiple comparisons between groups statistically analyzed (Fig. 4).
Fig. 1. Dose–response of hepatic PEPCK activity to AIA/DDC treatment. Animals were injected subcutaneously (sc) with three different
AIA doses and intraperitoneally (ip) with a single DDC dose, divided
into the following groups: group L, 100 mg AIA + 50 mg DDC/kg
body wt.; group M, 250 mg AIA + 50 mg DDC/kg body wt.; group H,
500 mg AIA + 50 mg DDC/kg body wt. Control group (group C) only
received vehicles: saline solution, sc and corn oil, ip. Each bar represents the mean ± S.E.M. of six animals. Activity is expressed as nmol
NADH oxidized/mg protein min. * P < 0.05 is significantly different
from group C; # P < 0.05 is significantly different from group L. Multiple comparisons among groups were performed using Tukey–Kramer’s
test.
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S.M. Lelli et al. / Toxicology 216 (2005) 49–58
Fig. 2. Dose–response of hepatic GP activity to AIA/DDC treatment.
Animals were injected with the same AIA and DDC doses indicated
in Fig. 1. Each bar represents the mean ± S.E.M. of six animals.
Activity is expressed as ␮mol Pi/g wet liver min. * P < 0.05 is significantly different from group C; # P < 0.05 is significantly different from
group L. Multiple comparisons among groups were performed using
Tukey–Kramer’s test.
3.4. Enzymatic defense systems
Fig. 5A and B shows SOD and CAT activities, respectively, measured in livers from animals treated with
AIA/DDC and in controls. SOD activity increased 235%
in group H, 41.6% in group M, and 19% in group L with
respect to the control group. On the contrary, CAT activity suffered 24, 11 and 9% inhibition in the same groups,
respectively. Both SOD and CAT showed a drug–dose-
Fig. 3. Dose–response of hepatic cytosolic carbonyl content to
AIA/DDC treatment. Animals were injected with the same doses
of AIA and DDC indicated in Fig. 1. Each bar represents the
mean ± S.E.M. of six animals. Content is expressed as nmol carbonyl/mg protein. * P < 0.05 is significantly different from group C;
# P < 0.05 is significantly different from group L. Multiple comparisons
among groups were performed using Tukey–Kramer’s test.
Fig. 4. Dose–response of hepatic lipid per oxidation to AIA/DDC treatment. Animals were injected with the same doses of AIA and DDC
indicated in Fig 1. Each bar represents the mean ± S.E.M. of six animals. Activity is expressed as nmol MDA/g wet liver h. * P < 0.05 is
significantly different from group C; # P < 0.05 is significantly different from group L; § P < 0.05 is significantly different from group M.
Multiple comparisons among treatment groups were performed using
Tukey–Kramer’s test.
dependent behavior in the multiple comparisons between
groups statistically analyzed.
4. Discussion
The hepatic acute porphyria model presented in this
study proved to be useful since it allows us to observe,
among other parameters indicative of porphyria, the significant increase in hepatic ALA concentration caused by
ALA-S de-repression. This de-repression is the result of
the depletion of the regulatory hepatic heme pool produced by the two drugs used in this study; AIA and DDC.
These porphyrinogenic drugs depleted the free regulatory heme pool, causing an imbalance. AIA acts through
heme alkylation, yielding “green pigments” that lead to
the suicidal and irreversible process of cytochrome P450 inactivation. In this process, the heme moiety of
cytochrome P-450 is destroyed and, consequently, the
free heme pool decreases (Ortiz de Montellano and Mico,
1981; Smith and De Matteis, 1980). In addition, DDC
leads to the formation of N-alkylporphyrins, which act
as inhibitors of ferrochelatase activity, thus reducing the
heme pool (Marks et al., 1988). Whiting and Granick
(1976) have demonstrated that, after 18 h, a combination of these drugs may result in a 600-fold increase of
ALA-S activity in chick embryo liver.
Although the animal model herein presented does not
exactly mimic any human hepatic porphyria, from the
biochemical point of view, it resembled quite accurately
the variegate porphyria. In fact, early reports on this
S.M. Lelli et al. / Toxicology 216 (2005) 49–58
Fig. 5. Dose–response of (A) SOD and (B) CAT activities to AIA/DDC
treatment. Animals were injected with the same doses of AIA and DDC
as indicated in Fig. 1. Each bar represents the mean ± S.E.M. of six animals. SOD activity is expressed as U/mg protein (in SOD assay, a unit is
defined as the amount of enzyme necessary to inhibit 50% epinephrine
autoxidation), and CAT activity as ␮mol H2 O2 consumed/mg protein
min. * P < 0.05 is significantly different from group C; # P < 0.05 is significantly different from group L; § P < 0.05 is significantly different
from group M. Multiple comparisons among treatment groups were
performed using Tukey–Kramer’s test.
human disease described an inhibition of ferrochelatase
activity (Cole and Marks, 1984), and later studies
reported an inhibition of hepatic protoporphyrinogenoxidase activity (Kappas et al., 1995).
The liver plays a major role in blood glucose homeostasis by maintaining a balance between the uptake
and storage of glucose via glycogenogenesis, and the
release of glucose via glycogenolysis and gluconeogenesis (Hers, 1990; Nordlie et al., 1999). In our study, the
activities of two enzymes were measured; one involved
in glucose synthesis (PEPCK) and the other in glycogen degradation (GP). Both activities appear particularly diminished. PEPCK showed deficient activity in
the model used by Correia and Lunetta (1989), in which
55
the administration of phenobarbital (a cytochrome P-450
hepatic inducer) and 3,5-dicarbethoxy-2,6-dimethy-4ethyl-1,4-dihydro-pyridine (a P-450 suicide inhibitor)
caused the abrupt depletion of hepatic heme. This model
presented a distinct pattern of accumulation of gluconeogenic intermediates proximal to PEPCK, such
as malate and aspartate, which increased significantly,
while all distal intermediates were definitely depleted
(Correia and Lunetta, 1989). PEPCK also exhibited
a lower activity in chronic intoxication with the porphyrinogenic drug HCB (Mazzetti et al., 2004). In this
case, the time-course effect on enzyme activity with this
xenobiotic showed an early decrease, which was intensified during the treatment. Stahl et al. (1993) also reported
a hepatic PEPCK decrease in rats treated with 2,3,7,8tetrachlorodibenzo-p-dioxin (another porphyrinogenic
drug) during 4 and 8 days due to a decrease in mRNA levels. As regards GP, there are not previous reports on the
determination of this activity on acute porphyria models. Taira et al. (2004) found that in a model of chronic
porphyria caused by hexachlorobenzene (HCB) administration to Wistar rats, this glycogenolytic enzyme also
exhibited a diminished activity during the 5, 7 and
9th weeks of treatment when compared to the control. Moreover, ultrastructural studies with HCB-treated
rats showed that glycogen replaced smooth endoplasmic reticulum proliferation (Mollenhauer et al., 1975).
It was also observed that numerous glycogen particles
appeared in the extensive cytoplasm area of hepatocytes
located in the intermediary zone, while only few monoparticles of glycogen were deposited in the centrilobullar
area (Böger et al., 1979). These reports prove that in the
HCB model there is an increase of hepatic glycogen due
to a decrease in GP activity.
PEPCK and GP decreases observed in the AIA/DDC
model herein presented agree with those reported for
other porphyrinogenic drugs, such as HCB and 2,3,7,8tetrachlorodibenzo-p-dioxin (Taira et al., 2004; Stahl
et al., 1993). According to these results, these decreases
observed in our model would lead to insufficient glucose
availability within the hepatocyte.
The increases in oxidative stress markers herein
reported – protein carbonyl content, TBARS, urinary
chemiluminescence and SOD antioxidant response –
agree with the oxidative damage caused by hepatic ALA
accumulation. In this respect, Bechara (1996) reported
that oxidative stress might be triggered by ALA in acute
intermittent porphyria and lead poisoning. Our work
showed that CAT suffered a slight but statistically significant decrease due to a reduction in the heme pool elicited
by both drugs. This was expected, considering the hemeprotein nature of the enzyme (Price et al., 1962).
56
S.M. Lelli et al. / Toxicology 216 (2005) 49–58
It is worth mentioning that both enzymatic responses
(PEPCK and GP), as well as the behavior of oxidative
stress markers already mentioned exhibited a similar pattern against the different AIA/DDC doses, suggesting
a link between ROS and enzyme decreases. However,
we can not discard other causes that could be concomitant and that could lead to a reduction in PEPCK and
GP activities as it was proposed for PEPCK decrease in
HCB-induced experimental porphyria. Such causes are
the effect of tryptophan (Mazzetti et al., 2004) and glucocorticoids (Lelli et al., 2004). In fact, it has already been
demonstrated that tryptophan administration resulted in
hypoglycaemia, that tryptophan and its metabolites alter
hepatic gluconeogenesis (Smith and Pogson, 1977), and
that hepatic tryptophan levels significantly increase in
HCB porphyric rats (Llambı́as et al., 2003). According
to Mazzetti et al. (2004), tryptophan would be responsible for the decrease in gluconeogenic PEPCK activity at
the higher stages of fungicide intoxication, thus adding
its effect to that exerted by the free radicals already mentioned.
It is worth mentioning that in an acute porphyria rat
model, in which heme was depleted by phenobarbital
and 3,5-dicarbethoxy-2,6-dimethy-4-ethyl-1,4-dihydropyridine treatment, Correia and Lunetta (1989) reported
a blockage in tryptophan degradation with the consequent increase in its hepatic level, concomitantly with a
blockage in gluconeogenesis.
On the other hand, and considering that glucocorticoids are important regulators of glucose synthesis, Lelli
et al. (2004) reported that HCB triggers an early hormonal disruption at the plasmatic glucocorticoids level,
as well as an alteration on the number and affinity of
its hepatic receptors in relation to PEPCK activity. They
postulate that this hormonal disturbance could be one
of the causes of the gluconeogenic blockage elicited by
HCB at the enzymatic level.
It is important to note the dose–response relationships observed both in protein carbonylation and in
PEPCK and GP enzymatic activities as a consequence of
AIA/DDC treatment. Based on these results it could be
speculated that the decrease of enzymatic activities could
be due, in part, to the oxidative damage suffered by their
protein structures during carbonylation. In addition, we
speculate that both enzymes (PEPCK and GP) would be
susceptible to carbonylation due to the presence of target amino acids in their sequences and especially in key
sites of their enzymatic functions. Thus, aminoacids sensitive to this kind of oxidative modification such as Arg,
Lys, and Thr, highly conserved in several species, can
be found in the active site of PEPCK (Aich et al., 2003).
On the other hand, carbonyl groups may be introduced
into proteins through a secondary reaction of the nucleophylic side chains of Cys, His, and Lys residues, with
aldehydes produced during lipid peroxidation (DalleDonne et al., 2003). The carbonylation of these critical
aminoacids of the active site would lead to a decrease
in enzymatic activity, in line with the results obtained
in our work. Assays on rat hepatic GP showed that one
Lys was responsible for enzymatic activity, and two Cys
were involved in the association of subunits that allow an
active protein conformation (Schiebel et al., 1992). On
the other hand, the lipid peroxidation increase observed
in this model also contributed to protein carbonylation
through ROS produced during that event.
Our results demonstrate for the first time that alterations caused by AIA/DDC on two key enzymes related
to glucose, one involved in its synthesis and the other in
its liberation, would lead to a deficit in the availability
of hepatic glucose in treated animals. Therefore, we can
speculate that these results could contribute to explain,
through the determination of gluconeogenic PEPCK and
glycogenolytic GP activities, the beneficial role of carbohydrate administration to patients suffering acute hepatic
porphyrias. In this type of diseases, the oxidative stress
caused by high ALA accumulation would act on the
protein structure of key enzymes of the carbohydrate
metabolism, diminishing the amount of hepatic glucose
available.
Acknowledgments
We thank Mrs. Carmen Aldonatti (Technical Assistance Career Member of CONICET) for her skilful technical assistance. This work was supported by grants from
the University of Buenos Aires (UBACyT) and CONICET. L.C. San Martı́n de Viale is a Scientific Research
Career Member of the CONICET (Consejo Nacional de
Investigaciones Cientı́ficas y Técnicas).
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