The toxicity and pharmacokinetics of dihydrosanguinarine in rat: A pilot study

Article abstract of DOI:10.1016/j.fct.2008.04.013

The quaternary benzo[c]phenanthridine alkaloid sanguinarine (SG) is the main component of Sangrovit, a natural livestock feed additive. Dihydrosanguinarine (DHSG) has recently been identified as a SG metabolite in rat. The conversion of SG to DHSG is a likely elimination pathway of SG in mammals. This study was conducted to evaluate the toxicity of DHSG in male Wistar rats at concentrations of 100 and 500 ppm DHSG in feed for 90 days (average doses of 14 and 58 mg DHSG/kg body weight/day). No significant alterations in body or organ weights, macroscopic details of organs, histopathology of liver, ileum, kidneys, tongue, heart or gingiva, clinical chemistry or hematology markers in blood in the DHSG-treated animals were found compared to controls. No lymphocyte DNA damage by Comet assay, formation of DNA adducts in liver by ³²P-postlabeling, modulation of cytochrome P450 1A1/2 or changes in oxidative stress parameters were found. Thus, repeated dosing of DHSG for 90 days at up to 500 ppm in the diet (i.e. approximately 58 mg/kg/day) showed no evidence of toxicity in contrast to results published in the literature. In parallel, DHSG pharmacokinetics was studied in rat after oral doses 9.1 or 91 mg/kg body weight. The results showed that DHSG undergoes enterohepatic cycling with maximum concentration in plasma at the first or second hour following application. DHSG is cleared from the body relatively quickly (its plasma levels drop to zero after 12 or 18 h, respectively).

Full text of DOI:10.1016/j.fct.2008.04.013

Food and Chemical Toxicology 46 (2008) 2546–2553
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
The toxicity and pharmacokinetics of dihydrosanguinarine in rat: A pilot study
b
c
d
a
Eva Vrublova a, Jitka Vostalova a, , Rostislav Vecera , Borivoj Klejdus , David Stejskal , Pavel Kosina ,
Adela Zdarilova a, Alena Svobodova a, Vaclav Lichnovsky e, Pavel Anzenbacher b, Zdenek Dvorak a,
Jaroslav Vicar a, Vilim Simanek a, Jitka Ulrichova a
*
a Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
b Department of Pharmacology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
c Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00 Brno, Czech Republic
d Department of Laboratory Medicine, Sternberk Hospital, 785 01 Sternberk, Czech Republic
e Department of Histology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
The quaternary benzo[c]phenanthridine alkaloid sanguinarine (SG) is the main component of SangrovitÒ,
a natural livestock feed additive. Dihydrosanguinarine (DHSG) has recently been identified as a SG
metabolite in rat. The conversion of SG to DHSG is a likely elimination pathway of SG in mammals. This
study was conducted to evaluate the toxicity of DHSG in male Wistar rats at concentrations of 100 and
500 ppm DHSG in feed for 90 days (average doses of 14 and 58 mg DHSG/kg body weight/day). No sig-
nificant alterations in body or organ weights, macroscopic details of organs, histopathology of liver,
ileum, kidneys, tongue, heart or gingiva, clinical chemistry or hematology markers in blood in the
DHSG-treated animals were found compared to controls. No lymphocyte DNA damage by Comet assay,
formation of DNA adducts in liver by 32P-postlabeling, modulation of cytochrome P450 1A1/2 or changes
in oxidative stress parameters were found. Thus, repeated dosing of DHSG for 90 days at up to 500 ppm in
the diet (i.e. approximately 58 mg/kg/day) showed no evidence of toxicity in contrast to results published
in the literature. In parallel, DHSG pharmacokinetics was studied in rat after oral doses 9.1 or 91 mg/kg
body weight. The results showed that DHSG undergoes enterohepatic cycling with maximum concentra-
tion in plasma at the first or second hour following application. DHSG is cleared from the body relatively
quickly (its plasma levels drop to zero after 12 or 18 h, respectively).
Article history:
Received 10 August 2007
Accepted 8 April 2008
Keywords:
Benzo[c]phenanthridine alkaloid
Dihydrosanguinarine
Sanguinarine
Dietary toxicity
Metabolism
Disposition
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and anti-inflammatory activities and as an active component in
the animal feed additive SangrovitÒ used for pigs and chickens
The quaternary benzo[c]phenanthridine alkaloid (QBA) san-
guinarine (SG) and its congener chelerythrine are elicitor-inducible
secondary metabolites (phytoalexines) with antifungal and
nematocidal activities in Caprifoliaceae, Fumariaceae, Meliaceae,
Papaveraceae and Rutaceae plants (Wink, 1999). In vitro studies
have shown that SG modulates a number of molecular targets in
the metabolism of mammalian cells (Malikova et al., 2006a,b). SG
is also used in toothpastes and mouthwashes for its antibacterial
(Dvorak et al., 2006a). On the other hand, the toxicity of the oil
of Argemone mexicana seeds has been attributed to SG and DHSG
which putatively initiate oxidative stress (ROS formation leading
to lipid peroxidation, depletion of GSH and decrease of total anti-
oxidant capacity) and death of red cell via formation of methemo-
globin (Das et al., 2005; Babu et al., 2006, 2007). Oxidative stress
(production of oxygen and nitrogen reactive species) is closely con-
nected with genotoxicity. In two previous studies we examined the
safety of feeding pigs (Kosina et al., 2004) and rats (Psotova et al.,
2006b) for 90 days with sanguiritrin, a mixture of SG and chel-
erythrine, extracted from Macleaya cordata. All animals remained
in good health over the duration of both experiments and mani-
fested no signs of the oxidative stress described for argemone oil
intoxication. SG-derived DNA adducts and/or DNA damage in livers
of pigs/rats exposed to sanguinarine were not detected. Recently
we found that the first step in SG metabolism in rat is the reduction
of the iminium bond leading to formation of DHSG (Vecera et al.,
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AOPP,
advanced oxidation protein products; AST, aspartate aminotransferase; AUC, area
under the plasma concentration–time curve; CYP, cytochrome P450; CHE, cholin-
esterase; DHSG, dihydrosanguinarine; GIT, gastrointestinal tract; GMT, gamma-
glutamyl transpeptidase; GPX, glutathione peroxidase; GSH, glutathione; SG,
sanguinarine; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive
substances.
* Corresponding author. Tel.: +420 585 632 316; fax: +420 585 632 302.
E-mail address: j.psotova@email.cz (J. Vostalova).
0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.04.013

E. Vrublova et al. / Food and Chemical Toxicology 46 (2008) 2546–2553
2547
other organs) was done. The urine was collected from the animal’s bladder (to pre-
vent urine components from oxidative processes and contamination by feces) and
analyzed for DHSG content. The blood was collected from the aortic bifurcation into
Na2EDTA-tubes (Sarstedt, Germany) and Heparin-Lithium-tubes (Sarstedt, Ger-
many). A part of the Na2EDTA blood was used for lymphocyte isolation for genotox-
icity assay and for blood count (0.5 ml). The rest of Na2EDTA blood was centrifuged
at 2500g for 10 min at 4 °C to obtain the plasma. The plasma aliquots were stored at
ꢁ80 °C for determination of DHSG/SG content and parameters of antioxidant capac-
ity. Heparin-lithium blood was centrifuged at 2500g for 10 min at 4 °C to obtain the
plasma for the clinical chemistry. Na2EDTA-erythrocytes were washed with phos-
phate-buffered saline and stored at ꢁ80 °C for determination of oxidative stress
parameters. Liver, muscle, kidney, heart, ileum, tongue, thymus, and brain were re-
moved, washed in cold phosphate-buffered saline and the selected organs were
weighed and stored at ꢁ80 °C for determination of DHSG/SG content. Livers were
utilized for DNA adduct formation analysis and parameters of oxidative stress
and were frozen and stored at ꢁ80 °C prior to analyses. Histological examination
was made of tongue, liver, ileum, kidney, gingiva and heart.
2007). We also found that SG and DHSG were completely elimi-
nated from plasma and liver after 24 h. We have never detected
the formation of the genotoxic benz[c]acridine (Psotova et al.,
2006a; Vecera et al., 2007), which had been suggested (Tandon
et al., 1993) as a product of SG transformation.
In contrast to SG, practically no information about DHSG biolog-
ical activity exists (Walterova et al., 1995). Only Vavreckova et al.
(1994) reported that DHSG was nontoxic in rat hepatocyte primary
cultures. The objective of this study was to assess the effects of 100
or 500 ppm DHSG in feed in a 90-day experiment in rats. The doses
(100 ppm and 5 ꢀ 100 ppm) were selected in order to relate this
study to that of Babu et al. 2006, who applied 1% of argemone oil
(i.e. 53 mg of DHSG/SG mixture) in one kg of feed (toxicity reported)
and to our previous experiments (Kosina et al., 2004; Psotova et al.,
2006b), where we added 100 mg of benzo[c]phenanthridine alka-
loids (SG/chelerythrine mixture) to 1 kg of feed (no toxicity found).
The duration of the study was selected as 90 days, a standard time
for subchronic toxicity. Moreover, farm animals consume feed with
additives containing a mixture of QBA, mainly sanguinarine maxi-
mally for 3 months. Evaluated in this study were: health status, body
weight, organ morphology, hematology, clinical chemistry parame-
ters, oxidative stress markers in blood and liver. Modulation of liver
microsomal cytochrome P450 and isoform 1A1/2 by DHSG and the
effect of DHSG on hepatocyte and lymphocyte DNA and its distribu-
tion in organs were also investigated. To corroborate the findings,
DHSG levels in plasma as well as the kinetics of DHSG elimination
were determined in a pharmacokinetic study using oral doses of
9.1 or 91 mg/kg body weight.
2.4. Determination of DHSG in diet, feces, urine, tissues and plasma
Diets and/or feces were extracted with acidified (1% HCl) methanol in a Soxhlet
extractor for 12 h and the extracts after appropriate dilution by mobile phase were
subjected to HPLC analysis.
Urine (0.4 ml) and/or plasma (0.4 ml) were mixed with methanol (0.6 ml or
0.8 ml, resp.), vortexed vigorously for 1 min and centrifuged at 12,000g for
15 min at 4 °C. The supernatant (30 ll) was applied to an HPLC column.
Tissues: Liver, kidney, muscle, ileum, tongue, brain and heart (1 g) were homog-
enized with a mechanical homogenizer in 4 ml of a 0.01 M 1-heptanesulfonic acid
in 95% acetonitrile. The homogenate was vortexed vigorously for 1 min, and centri-
fuged at 12,000g for 15 min at 4 °C. After evaporation of supernatant (3 ml) under
N2 at 50 °C, it was dissolved in methanol (0.5 ml). The sample was then filtered
through a 0.45 lm Teflon filter and applied (30 ll) to a HPLC column.
High performance liquid chromatography separation and mass spectrometry
detection and identification of SG and DHSG were carried out as described else-
where (Psotova et al., 2006a; Vecera et al., 2007).
2. Materials and methods
2.5. Clinical chemistry
2.1. Test substance
On day 90, following sacrifice, blood plasma analyses of sodium, potassium,
chlorides, bilirubin, cholesterol, urea, creatinine, alanine aminotransferase (ALT),
aspartate aminotransferase (AST), gamma-glutamyl transferase (GMT), alkaline
phosphatase (ALP), cholinesterase (CHE), total protein and prealbumin were ana-
lyzed on an Advia analyser 1650 (Bayer, USA).
Dihydrosanguinarine (DHSG), 99% purity, MP 189–191 °C was prepared from
sanguinarine by reaction with NaBH4 in methanol (Brossi and Borer, 1965).
Benz[c]acridine was supplied by Fluka (Buchs, Switzerland) and sanguinarine from
Sigma (USA).
2.6. Hematology
2.2. Diets
Hemoglobin, hematocrit, erythrocytes, mean erythrocyte volume, thrombo-
cytes, leukocytes and differential leukocyte count were analyzed in Na2EDTA blood.
Experimental diets: DHSG (100 mg or 500 mg) was blended with the powdered
commercial diet (800 g), microcrystalline cellulose (190 g) and magnesium stearate
(10 g) to prepare the feed pellets.
Control diet: The powdered commercial diet (800 g) was blended with micro-
crystalline cellulose (190 g) and magnesium stearate (10 g).
2.7. Parameters of oxidative stress
The diets were prepared monthly and analyzed by HPLC periodically to confirm
concentration, homogeneity and stability of DHSG in the diet. The pellets were
stored in paper bags and kept dry.
Lipid peroxidation was assessed by measuring the presence of thiobarbituric
acid reactive substances (TBARS) in the plasma, erythrocytes or liver homogenates
(Buege and Aust, 1978). The level of glutathione (GSH) in erythrocytes and liver
homogenate was determined according to Sedlak and Lindsay (1968) using Ellman’s
reagent. The plasma level of total SH-group was determined according to Hu (1994).
An indirect spectrophotometric method used for assessment of superoxide dismu-
tase (SOD) activity in erythrocytes and liver homogenate was based on the genera-
2.3. Animals
The study was approved by the Ethics Committee, Ministry of Education, Czech
Republic and conducted in compliance with the Experimental Animals Protection
Act No. 167/1993 L.C. Male Wistar rats (n = 18; 210 10 g bw) were purchased from
BioTest Ltd., Konarovice, Czech Republic. The rats were acclimatized 1 week before
the experiment. They were kept in plastic cages containing dust-free sawdust, two
animals per cage. On the day of treatment, the animals were 8 weeks old with a
mean body weight SD of 240 8 g (n = 18). They were randomized to three groups
(6/group); control – Group 1 (241 11 g bw); Group 2, 100 ppm DHSG (236 7 g
bw); Group 3, 500 ppm DHSG (243 5 g bw). Group sizes of six were considered
acceptable for this pilot investigation of toxicity.
During the acclimatization period and during testing, the conditions in the ani-
mal room were as follows: temperature (23 2 °C; checked daily); relative humid-
ity (30–70%); light/dark cycle 12 h/12 h. The animals had free access to their
respective diets and water. The feed consumption was checked twice a week. The
health of the animals was checked daily and body weights were monitored twice
a week and prior to sacrifice.
tion of OÅ2ꢁ by
a mixture of nitro blue tetrazolium, NADH and phenazine
methosulfate (Ewing and Janero, 1995). Glutathione peroxidase (GPX) activity in
erythrocytes and liver homogenate was assayed by a modification of the method
of Tappel (1978). Catalase activity in erythrocytes and liver homogenate was deter-
mined according to Beers and Sizer (1952) a method based on measuring the quan-
tity of decomposed hydrogen peroxide in a unit of time. Advanced oxidation protein
products (AOPP) in plasma were assessed spectrophotometrically at 340 nm. The
plasma and liver homogenate total antioxidant capacity (TAC) was measured by
cyclic voltammetry. The protein concentration was determined by the Bradford
method.
2.8. Total and specific CYP1A1/2 content of cytochrome P450
Liver microsomes were isolated by ultracentrifugation and the cytochrome
P450 content was measured according to Omura and Sato (1964). The microsomal
protein concentration was measured according to Lowry. The levels of cytochromes
P450 1A1/2 were assessed in pooled microsomal samples by immunoblotting after
Administration of DHSG: The animals consumed ad libitum either the standard
diet or the diets containing 100 or 500 ppm DHSG, for 90 days.
Sample collection and preparation: The animals were deprived of food 12 h be-
fore terminal i.m. anesthesia by fentanyl (4 lg/100 g bw), medetomidin (20 lg/
100 g bw) and diazepam (0.5 mg/100 g bw). After opening the abdominal cavity,
macroscopic examination of the main organs (GIT, heart, kidneys, liver, lungs and
SDS–PAGE electrophoresis using
a primary antibody against human CYP1A1
(CYP1A1 (G-18) goat polyclonal IgG; dilution 1/500) purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, USA) and a secondary antibody conjugated with
horseradish peroxidase using chemiluminescence detection.

2548
E. Vrublova et al. / Food and Chemical Toxicology 46 (2008) 2546–2553
2.9. Histopathology
age doses over 90 days were 14 (Group 2) and 58 mg/kg/day
(Group 3), respectively. No significant alteration in feed consump-
tion or health status was found in any group.
Liver, ileum, kidney, tongue, heart and gingiva specimens were fixed in Baker
mixture, embedded in paraffin and 7 lm thick sections were cut on a rotary micro-
tome. Sets of histological sections were stained with hematoxylin-eosin and PAS.
The histological evaluation was performed on an Olympus BX 40 light microscope.
3.3. Body and organ weight
2.10. Genotoxicity
The weight gain curves displayed very similar courses for
Groups 1 and 2 (Fig. 1). There was a slightly greater weight in-
crease in animals of Group 3 compared to Groups 1 or 2 and this
difference was significant (p < 0.05), as evidenced by comparison
of regression coefficients for the gain weight curves (Fig. 1).
There was no significant difference in weights of liver, right and
left kidney, heart, brain and thymus for any of the groups of ani-
mals (Table 1).
Detection of single-stranded DNA breaks: The DNA breaks were measured in
peripheral lymphocytes using an alkaline version of Comet assay (Vodicka et al.,
2001). Lymphocytes were isolated on Histopaque 1077 gradients. DNA damage
was analyzed after staining by ethidium bromide on an Olympus IX 70 fluorescence
microscope. One hundred cells per slide were analyzed and divided into four classes
of DNA damage standard scale. Total DNA damage was calculated: (number of cells
in class 1 ꢀ 1 + number of cells in class 2 ꢀ 2 + number of cells in class 3 ꢀ 3 + num-
ber of cells in class 4 ꢀ 4)/100. The maximal damage was given a value of 400.
Analysis of DNA adducts in liver: DNA was isolated from liver tissues of rats by
the phenol/chloroform procedure and the 32P-postlabeling assay was performed
as described previously (Stiborova et al., 2002). The detection limit of relative ad-
duct labeling was 0.1/108 nucleotides.
3.4. Clinical chemistry parameters
The following parameters were investigated: sodium, potas-
sium, chloride, bilirubin, urea, creatinine, cholesterol, ALT, AST,
GMT, ALP, CHE, total protein and prealbumin (Table 2). Except
for a significant decrease in ALT activity in Group 2, none of the
tested parameters showed any significant difference between
DHSG-treated and control animals.
2.11. Pharmacokinetics of DHSG
The animals were starved for 10 h before gavage administration of DHSG. In to-
tal, 132 male Wistar rats (260 10 g bw) were used in the pharmacokinetic study. If
not stated, the conditions were the same as for the oral toxicity experiment. The ga-
vage administration was used as an acceptable route in oral toxicity studies. The
difference between the intragastric route (gavage) and the dietary route should
not have been insignificant as most absorption takes place in the stomach and
the GIT. Since there was no information on the pharmacokinetics of DHSG, we chose
36 h to increase the chances of detecting metabolites. The rats were given a single
oral dose of DHSG suspension (9.1 or 91 mg/kg bw) in 1.0 ml water. After 0.5, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 28, 32 and 36 h, three rats were ter-
minally narcotized i.m. anesthesia by fentanyl (4 lg/100 g bw), medetomidin
(20 lg/100 g bw) and diazepam (0.5 mg/100 g bw). Urine were collected at 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 28, 32 and 36 h intervals. After
opening the abdominal cavity, a macroscopic examination of the main organs, GIT,
heart, kidney, liver and lungs was performed. The blood was collected from the aor-
tic bifurcation into Na2EDTA (1 mg/ml) and centrifuged at 10 °C and 2500g for
10 min to obtain plasma. The plasma aliquots and the liver samples washed with
phosphate-buffered saline were stored at ꢁ80 °C for the determination of DHSG
content. Experimental data were derived from three animals per time and dose.
Interruption of the enterohepatic cycle of DHSG: Immediately after oral applica-
tion of DHSG (91 mg/kg bw) the rats were narcotized i.m. anesthesia by fentanyl
(4 lg/100 g bw), medetomidin (20 lg/100 g bw) and diazepam (0.5 mg/100 g bw).
The billiary duct was ligated and after 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 9 h the blood
was collected from the aortic bifurcation into Na2EDTA (1 mg/ml) and centrifuged
at 10 °C and 2500g for 10 min to obtain plasma. At the end of the experiment, mac-
roscopic examination was done of the main organs, GI-tract, heart, kidneys, liver
and lungs. The plasma aliquots and phosphate-buffered saline washed liver were
stored at ꢁ80 °C for the determination of DHSG content. Experimental data were
derived from three animals.
600
500
400
Control
100 ppm DHSG
300
500 ppm DHSG
200
0
20
40
60
80
100
Time (day)
Fig. 1. Effect of dihydrosanguinarine on body weight of rats. The animals consumed
ad libitum either standard diet (Group 1) or diets containing 100 ppm (Group 2) or
500 ppm (Group 3) DHSG, for 90 days as described in Section 2. Body weights were
monitored twice a week and prior to sacrifice. *Body weight of animals in Group 3
was significantly different from that of Group 1 and 2 (p < 0.05).
Table 1
2.12. Statistical analyses
Rat body and organ weights on day 90
All values were expressed as mean SD and ANOVA was used to analyze data
(p < 0.05). A regression analysis (logarithmic regression model) was used to test
the significance of differences in gain weight in the three groups of rats (p < 0.05).
Weight (g)
Groups 1–3
DHSG dose level (mg/kg/day)
0
14
511 23
58
553 64
Body
Liver
526 29
3. Results
12.75 1.58
1.63 0.33
1.68 0.32
1.38 0.18
0.41 0.09
2.00 0.13
12.17 0.96
1.63 0.21
1.61 0.17
1.42 0.08
0.40 0.16
1.95 0.12
13.35 1.75
1.69 0.18
1.66 0.16
1.42 0.19
0.44 0.10
2.05 0.10
Right kidney
Left kidney
Heart
Thymus
Brain
3.1. Diet analysis
The concentration, homogeneity and stability of DHSG in
the prepared diets were periodically analyzed by HPLC. The DHSG
concentration in diets was 97.5 7.3 lg/g (Group 2) and
478.1 20.6 lg/g (Group 3), respectively.
Organ weight/body weight (%)
Liver
2.42 0.20
2.39 0.26
0.32 0.03
0.32 0.02
0.28 0.02
0.08 0.03
0.38 0.02
2.41 0.14
0.31 0.03
0.30 0.02
0.26 0.04
0.08 0.01
0.38 0.05
Right kidney
Left kidney
Heart
Thymus
Brain
0.31 0.06
0.32 0.06
0.26 0.02
0.08 0.02
0.38 0.03
3.2. Feed consumption
The daily diet consumption was 40 g on average. Daily dose of
DHSG for 100 ppm and 500 ppm (Groups 2 and 3), respectively, fell
from 17 (Group 2) and 80.8 mg/kg/day (Group 3) at the beginning
to 7.83 and 35.5 mg/kg/day at the end of the experiment, i.e. aver-
The animals were sacrificed on day 90 as described in Section 2. Body and organ
weights were determined and are expressed as mean SD of six animals per group.
An organ weights are normalized for body weight (lower part of Table).

E. Vrublova et al. / Food and Chemical Toxicology 46 (2008) 2546–2553
2549
Table 2
Table 4
Clinical chemistry parameters in plasma
Oxidative stress parameters in plasma, erythrocytes and liver
Parameter
Unit
Groups 1–3
Parameter
Unit
Group 1–3
DHSG dose level (mg/kg/day)
DHSG dose level (mg/kg/day)
0
14
58
0
14
58
Sodium
Potassium
Chloride
Bilirubin
Cholesterol
Urea
Creatinine
ALT
AST
GMT
ALP
CHE
Total protein
Prealbumin
mmol/l
mmol/l
mmol/l
lmol/l
lmol/l
mmol/l
lmol/l
lkat/l
lkat/l
lkat/l
lkat/l
lkat/l
g/l
146.0 1.9
3.85 1.00
95.7 1.6
1.57 0.28
1.43 0.23
6.02 1.66
54.5 5.1
1.03 0.11
1.50 0.15
0.03 0.00
1.28 0.29
5.7 0.8
146.0 2.6
3.50 0.10
96.8 1.0
1.78 0.40
1.70 0.41
6.60 1.36
54.0 7.3
145.2 1.9
3.97 0.76
95.2 1.2
1.42 0.41
1.38 0.27
6.52 1.35
58.5 4.5
0.90 0.13
1.64 0.16
0.03 0.00
1.16 0.15
5.5 0.5
TACa
nA/gd
lmol/l
18.54 3.69
35.85 6.13
2.56 0.46
376.4 33.1
10.06 2.15
379.2 62.8
165.4 29.9
30.84 6.96
1.17 0.28
19.61 7.10
33.30 3.44
2.74 0.44
397.9 25.3
9.99 1.18
419.3 59.7
148.1 25.9
28.64 3.09
1.11 0.17
22.13 3.8
40.37 7.4
2.79 0.5
406.3 37.4
10.58 2.05
417.0 64.7
142.2 29.9
30.42 4.0
0.92 0.1
AOPPa
SH-groupsa
TBARSa
GSHb
mmol/gd
nmol/gd
lmol/ge
nmol/ge
lmol/min/ge
lmol/min/ge
U/ge
TBARSb
GPXb
0.76 0.13*
1.43 0.29
0.03 0.00
1.13 0.50
5.5 1.9
Catalaseb
SODb
TACc
lA/gd
0.135 0.025
16.70 1.50
150.4 10.8
0.140 0.024 0.150 0.02
GSHc
lmol/gd
nmol/gd
16.50 0.99
158.0 23.0
264.1 34.6
29.44 3.9
2.54 0.46
0.33 0.07
17.37 2.3
153.3 15.8
220.8 37.2
26.79 5.2
2.92 0.8
TBARSc
GPXc
73.0 2.2
0.062 0.017
69.3 5.0
0.058 0.010
69.5 1.8
0.070 0.026
lmol/min/gd 263.4 16.2
lmol/min/gd 29.80 5.01
g/l
Catalasec
SODc
U/gd
2.74 0.57
0.36 0.05
Cytochrome P450c lmol/gd
0.39 0.09
On day 90 the animals were sacrificed. The following parameters listed in the table
were investigated.
*
The value significantly different from control group (p < 0.05).
On day 90 the animals were sacrificed. The parameters listed in the table were
determined.
a
Plasma.
Erythrocytes.
Liver.
Values are expressed per 1 g of protein.
Values are expressed per 1 g of hemoglobin.
b
3.5. Hematological parameters
c
d
e
No effect of DHSG was found on monitored hematological
parameters, i.e. hemoglobin, hematocrit, erythrocytes, mean eryth-
rocyte volume, thrombocytes, leukocytes or differential leukocyte
count (Table 3).
Group
2
BNF
1
3
3.6. Oxidative stress parameters and content of cytochrome P450
We found no significant effect of DHSG on the selected param-
eters of oxidative stress in plasma, erythrocytes or liver. In addi-
tion, no alterations in level of total liver cytochrome P450 were
found (Table 4). We also determined levels of CYP1A1/2 proteins
in pooled samples of rat liver microsomes for each experimental
group. We found no change in CYP1A1/2 protein levels in any of
the three groups of animals, which is a positive finding from the
point of view of chemically induced carcinogenesis (Fig. 2).
Fig. 2. Effect of dihydrosanguinarine on CYP1A1/2 protein levels. Hepatic micro-
somes were isolated after animal sacrifice as described in Section 2. Samples were
pooled (i.e. six samples in each experimental group) and analyzed by immuno-
blotting. Western blot analysis of CYP1A1/2 proteins is shown.
Table 5
Effect of DHSG on DNA in peripheral lymphocytes by Comet assay
Parameter
Group 1–3
3.7. Genotoxicity
DHSG dose level (mg/kg/day)
0
14
58
To examine the genotoxicity of DHSG, two different methods
were used. First, the formation of single-stranded DNA breaks in
peripheral lymphocytes was analyzed by Comet assay. In all three
groups of animals, no significant differences were found (Table 5).
Next we examined the effect of DHSG on the formation of DNA
adducts in vivo on samples of DNA isolated from rat livers, both
those exposed to DHSG and the control group. The 32P-postlabeling
assays were carried out (Stiborova et al., 2002), no DNA adducts in
Damaged lymphocytes
14.42 16.30
17.58 14.29
12.92 5.88
At day 90 the animals were sacrificed, the peripheral lymphocytes were isolated
and Comet assays were performed as described in Section 2.
the livers of all three animal groups were detected (data not
shown).
3.8. Microstructure of tongue, heart, ileum, gingiva, kidney and liver
Tongue: Compared to the control, there were minor changes in
Group 2, specifically a subtle increase in the number of lympho-
cytes in mucosal papillae and a slightly increased number of mito-
ses in sites without papillae. A similar effect of DHSG was observed
on the tongue, i.e. a diminution of lymphocytes in the papillae and
a slightly increased number of mitoses in sites without papillae in
Group 3.
Heart: Heart was free of pathological findings in both DHSG-
treated groups of animals. In Group 2 there was only vasodilatation
of some coronary arteries. The heart muscle tissue was free of
pathological findings.
Table 3
Hematology parameters
Parameter
Unit
Groups 1–3
DHSG dose level (mg/kg/day)
0
14
0.455 0.02 0.458 0.02
159.2 5.5 162.3 7.6
8.9 0.44 9.008 0.3
58
Hematocrit
l
g/l
tera/l
fl
0.45 0.04
Hemoglobin
Erythrocytes
Mean volume of
erythrocytes
Leukocytes
159.8 10.2
9.1 0.87
49.7 1.5
51.0 2.2
51.0 2.5
giga/l
giga/l
5.38 1.23
5.02 1.18
6.40 1.63
Thrombocytes
832.5 114.1 881.3 44.7 895.7 103.0
Ileum: Compared to Group 1 (control), in Group 2 there were
tiny foci of edema in the mucosal connective tissue closely under
the epithelium in the proximal part of ileum villi. There was no evi-
On day 90 the animals were sacrificed. The parameters listed in the table were
determined.

2550
E. Vrublova et al. / Food and Chemical Toxicology 46 (2008) 2546–2553
Fig. 3. Representative microphotographs of the liver and ileum of the rat from Group 1 and 3. Magnification 60ꢀ. Liver (A) and ileum (C) of the control rat, liver (B) and ileum
(D) of the DHSG (500 ppm)-treated rat.
Table 6
25
20
15
10
5
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
A
Determination of DHSG/SG in plasma and organs
Sample
DHSG/SG (ng/g)a
Group 2
Group 3
Plasmab
Liver
Kidney
Ileum
Muscle
Heart
Tongue
Brain
nd/nd
0.50 0.13c/nd
0.56 0.33/1.34 1.68
1.77 0.47/nd
84.79 36.11/nd
0.08 0.06/nd
0.31 0.20/nd
5.16 2.46/nd
0.21 0.08/nd
0.21 0.22/0.19 0.12
0.32 0.26/nd
16.70 4.80/nd
0.02 0.03/nd
0.05 0.05/nd
0.84 0.56/nd
0.04 0.02/nd
0
0
10
20
30
40
Time (hrs)
a
Detection limit of DHSG in one gram of tissue was 178 fg, that of SG was 358 fg.
ng/ml of plasma.
A proportional decrease of the concentration with time of starvation during
b
c
0
10
20
30
40
Time (hrs)
plasma collection was observed (0.71–0.38 ng/ml); nd – not determined.
160
35
30
25
20
15
10
5
B
140
120
100
80
dence of the infiltrate in any of the specimens. The ilea of animals
in Group 3 displayed a picture similar to Group 2.
Gingiva: The gingiva of all animals in Groups 1–3 was free of
pathological findings.
Kidney: In Groups 2 and 3 there were minor foci of hyperemia
and capillary dilatation.
60
40
20
0
Liver: In the liver of animals of Group 3 there were foci of micr-
ovesicular steatosis in hepatocytes and very sporadic tiny foci of
infiltrate formed by polymorphonuclears in the portobilliary
spaces. In other specimens no pathological alterations were found.
Selected microphotographs of the liver and ileum of the rat of
Groups 1 and 3 are shown in Fig. 3.
0
10
20
30
40
Time (hrs)
0
0
10
20
30
40
Time (hrs)
In all studied tissues (Group 2 and 3) morphological findings
corresponded to those of control animals (Group 1).
Fig. 4. Time course of dihydrosanguinarine in plasma and liver. Concentration pr-
ofiles of DHSG (ng/ml) in rat blood plasma (ꢂ) and liver (s) after a single intragastric
administration of DHSG (two doses, 9.1 mg/kg bw (A) or 91 mg/kg bw (B)). Expe-
rimental conditions are described in Section 2.
3.9. Distribution of alkaloids
At the end of the 90-day experiment, in Group 2 and 3, feces, ur-
ine, plasma, liver, kidney, muscle, brain, tongue and heart were
analyzed for the DHSG/SG content (Table 6). Both compounds were
found in feces and liver of Groups 2 and 3. Quantities of DHSG/SG
in feces were 109.0 6.8/19.7 5.3 lg/g (Group 2) and

E. Vrublova et al. / Food and Chemical Toxicology 46 (2008) 2546–2553
2551
704.3 22.5/127.4 0.6 lg/g (Group 3). In all other body tissues of
Groups 2 and 3 and the plasma of Group 3 DHSG only was deter-
mined. No detectable SG/DHSG quantities in urine were found,
which confirmed the results of the pharmacokinetic experiment
evidencing a rapid elimination of DHSG from the organism.
ognized in the corresponding curve at 1, 5, 7, 10 and 14 h (Fig. 4B).
A plasma concentration–time course like this or similar is often
indicative of an enterohepatic circulation showing multiple peaks
and an extended half-life of the respective substance (Rang et al.,
2001). In parallel, the liver DHSG content was also determined,
and exhibited three to four maxima as well (see inserts in Fig. 4A
and B).
A DHSG enterohepatic circulation was confirmed using rats
with ligature of the biliary duct. The time course for levels of DHSG
in plasma after a single intragastric administration (91 mg/kg) is
shown in Fig. 5. DHSG was absorbed relatively slowly since the
maximum plasma level was observed during the sixth sampling
time (i.e. 5 h after administration). However, no multiple peaks
were observed suggesting that ligation of the biliary duct had
interrupted cycling.
3.10. Determination of the pharmacokinetic parameters of
dihydrosanguinarine (DHSG) in rat
In the final part of the experiment, we did a pharmacokinetic
study. The time course for levels of DHSG (ng/ml) in plasma after
a single intragastric administration dose of 9.1 or 91 mg/kg bw
are shown in Fig. 4. DHSG was absorbed relatively rapidly. The
plasma level peaked at the third sampling time (i.e. 2 h after
administration for 9.1 mg/kg) and the second sampling time (i.e.
1 h after administration for 91 mg/kg). The time course for levels
of DHSG in rat plasma showed for both doses multiple peaks sug-
gesting the enterohepatic circulation of DHSG. For the lower dose,
three peaks were observed at 2, 7 and 10 h after DHSG administra-
tion (Fig. 4A); with the higher dose, up to five peaks could be rec-
The calculated values of cmax, tmax, and of the AUC0?1 for DHSG
in plasma are given in Table 7. The data show that the AUC0?1 as
well as the cmax values increase in rat plasma with the DHSG higher
dose; comparison of data for lower and higher doses of DHSG indi-
cate that the overall balance of administered substance was
maintained.
10
8
4. Discussion
The higher dose of DHSG (58 mg/kg/day) resulted in a small (5%
vs. control) increase in body weight gain after 3 months which
reached statistical significance. Despite the small magnitude of this
change and the small sample size (n = 6), the finding was consid-
ered as giving some support to the current use of QBA-containing
additives as livestock feed stimulant.
6
4
2
0
0
2
4
6
8
10
DHSG in daily doses of 14 or 58 mg/kg over 3 months in rats had
no significant effect on any of the parameters tested (Tables 1–5).
However, long-term administration of 58 mg DHSG/kg/day (Group
3) caused a DHSG accumulation in various tissues (Table 6). Tissue
DHSG concentration corresponded well with its intake from the
feed (feed content 58 mg/kg/day, Group 3 vs. 14 mg/kg/day, Group
2). The tissues that contained the higher level of DHSG were those
in contact with passage of the diet (tongue, ileum). A smaller quan-
tity of SG together with DHSG was found in liver (Table 6). We cur-
rently have no plausible explanation for the SG presence in liver.
SG/DHSG in ratio 1:5.7 were further found in feces. The presence
of SG in feces may have been due to the metabolic activity of intes-
tinal microflora. In contrast to feces, we found no detectable SG/
DHSG quantities in urine (collected under experimental conditions
used). No benz[c]acridine was found in any sample analyzed in this
study, making questionable any explanation of DHSG toxicity (Das
Time (hrs)
Fig. 5. Time course of dihydrosanguinarine in plasma with a ligature of the billiary
duct. Concentration profile of DHSG (ng/ml) in rat blood plasma after a single int-
ragastric administration of DHSG (91 mg/kg bw) using experimental animals with a
ligature of the biliary duct. Experimental conditions are described in Section 2.
Table 7
Pharmacokinetic parameters of DHSG
Dose level (ng/kg bw)
9.1
91
91(ligature)
tmax (h)
2.0
1.69
1.0
28.08
51.86 2.05
5.0
7.66
cmax (ng/ml)
AUC0?1 (mg/ml h)
9.88 0.36
38.46 0.48
O
O
O
O
H+
A
A
B
B
A
B
O
O
OH-
+
C
6
D
7
O
C
D
7
O
C
6
H
D
7
O
8
8
8
N
N
N
SG per os
CH
CH
CH
O
O
O
3
3
3
6
-
H
Cl
OH
SANGUINARINE (SG)
SG PSEUDOBASE
DIHYDROSANGUINARINE
ELIMINATION
N
BENZ[c]ACRIDINE
Fig. 6. In vivo transformations of sanguinarine.

2552
E. Vrublova et al. / Food and Chemical Toxicology 46 (2008) 2546–2553
et al., 2005) based on the assumption of benz[c]acridine formation.
No histopathological changes in any of organs examined (Fig. 3)
were observed.
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