Relationship of hepatotoxicity and the target tissue dose of decabrominated diphenyl ether in subacutely exposed Wistar rats

Background/Aim. Based on numerous studies in animals, the most prominent toxic effects of decabrominated diphenyl ether (BDE-209) are observed in the liver, thyroid hormone homeostasis, reproductive and nervous systems. BDE-209 exhibits its toxic effects partly through the aryl hydrocarbon (Ah) receptor and consequent induction of hepatic microsomal enzymes. The aim of this study was to assess the hepatotoxic effect vs target tissue dose of BDE209 in the subacutely orally exposed Wistar rats. Methods. Effects were examined on male Wistar rats, weighing 200– 240 g, exposed to doses of 1,000, 2,000 or 4,000 mg BDE209/kg body weight (bw)/day by gavage during 28 days. Animals were treated according to the decision of the Ethics Committee of the Military Medical Academy, No 96671/2011. Evaluation of the hepatotoxic effect was based on: relative liver weight water and food intake, biochemical parameters of liver function [aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gama glutamyl transferase (γ-GT)], and oxidative stress parameters in liver homogenates [malondialdehiyde (MDA), superoxide dismutase (SOD), -SH] and morphological and pathohistological changes in the liver. For the assessment of internal dose – response relationship, lower confidence limit of Benchmark dose (BMDL) of 5% or 10% i.e. BMDL5 or BMDL10, were calculated using PROAST software. Results. After the application of 1,000, 2,000 or 4,000 mg BDE-209/kg bw/day, the concentrations of BDE-209 measured in liver were 0.269, 0.569 and 0.859 mg/kg of liver wet weight, (ww) respectively. Internal doses correlated with external (r = 0.972; p < 0.05) according to equation: internal dose (mg BDE-209/kg of liver ww) = 0.0002  external dose (mg/kg bw/day) + 0.0622. Hepatotoxicity was demonstrated based on significant increase in AST and γ-GT activities and the degree of histopathological changes. The lowest BMDL5 of 0.07228 mg BDE-209/kg of liver ww, correlating to external dose of 39 mg/kg/day, indicated the increase of AST activity as the most sensitive biomarker of BDE-209 hepatotoxicity in subacutely exposed rats. Conclusion. The results of the present work add up to the issue of BDE-209 toxicity profile with a focus on relationship between internal dose and hepatotoxicity. Critical internal dose for the effect on AST of 0.07 mg/kg of liver ww, corresponding to external dose of 39 mg/kg/day, is the lowest dose ever observed among the studies on BDE-209 hepatotoxicity. For the persistent substances with low absorption rate such as BDE-209, critical effect based on internal dose in majority of cases is considered as more precisely defined than the effect established based on external dose, particularly.

PROAST software.Results.After the application of 1,000, 2,000 or 4,000 mg BDE-209/kg bw/day, the concentrations of BDE-209 measured in liver were 0.269, 0.569 and 0.859 mg/kg of liver wet weight, (ww) respectively.Internal doses correlated with external (r = 0.972; p < 0.05) according to equation: internal dose (mg BDE-209/kg of liver ww) = 0.0002  external dose (mg/kg bw/day) + 0.0622.Hepatotoxicity was demonstrated based on significant increase in AST and γ-GT activities and the degree of histopathological changes.The lowest BMDL 5 of 0.07228 mg BDE-209/kg of liver ww, correlating to external dose of 39 mg/kg/day, indicated the increase of AST activity as the most sensitive biomarker of BDE-209 hepatotoxicity in subacutely exposed rats.Conclusion.The results of the present work add up to the issue of BDE-209 toxicity profile with a focus on relationship between internal dose and hepatotoxicity.Critical internal dose for the effect on AST of 0.07 mg/kg of liver ww, corresponding to external dose of 39 mg/kg/day, is the lowest dose ever observed among the studies on BDE-209 hepatotoxicity.For the persistent substances with low absorption rate such as BDE-209, critical effect based on internal dose in majority of cases is considered as more precisely defined than the effect established based on external dose, particularly.

Introduction
Polybrominated diphenyl ethers (PBDEs) are considered to be extremely efficient flame retardants 1,2 .Decabrominated diphenyl ether (BDE-209) is a major component of deca-BDE commercial mixture [3][4][5][6] that can migrate from the product, get into the environment and pose a risk for the human health.The main source of exposure to BDE-209 is from the diet, however inhaled air could also contribute to its entire body burden 7,8 .After entering the organism, BDE-209 exhibits its toxic effects partly through the aryl hydrocarbon (Ah) receptor and consequent induction of hepatic microsomal enzymes.It may also induce production of reactive oxygen species 7 found to be related to DNA damage.Based on numerous studies [8][9][10][11][12][13][14][15][16][17] on animals, the most prominent toxic effects of PBDEs are observed in the liver, thyroid hormone homeostasis, reproductive and nervous systems.
While the liver is one of the main target organs of its toxicity, the aim of this study was to assess the hepatotoxic effect of BDE-209 in the subacutely orally exposed Wistar rats.Considering that BDE-209 has poor solubility the level of its absorption and the amount reaching target tissues are difficult to predict.We assumed that the dose measured at target site is more appropriate than orally given dose, allowing us to more accurately perceive the liver changes from the relationship of internal dose and intensity of effects.Evaluation of hepatotoxic effects was based on serum liver enzymes activity: aspartat aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (γ-GT), alkaline phosphatase (ALP), degree of histopathological changes, as well as oxidative stress parameters in liver homogenates: malondialdehyde (MDA), activity of total superoxide dismutase (SOD) and content of sulfhydryl (-SH) groups.

Experimental animals
Male albino Wistar rats, weighing 200 g to 240 g were obtained from a disease-free stock bred at the Military Medical Academy in Belgrade, Serbia.The animals were housed in plastic cages with wire mesh top, in a climate-controlled facility with a constant 12-hour day and night cycle at 20°C to 24°C and relative humidity between 40% and 60%.The animals had free access to food and water throughout the study and were treated according to the guidelines for animal studies (No. 9667-1/2011), issued by the Academy's Ethical Committee.

Experimental protocol
After a quarantine period of 14 days, each group of animals (n = 8 per group) was receiving treatment solution by gavage in a volume of 0.5 mL/kg body weight (bw) day for 28 days.Control animals were receiving water (the control group), while rats in the vehicle control group were receiving DMSO alone the (DMSO group).Three groups were receiving BDE-209 as a suspension in DMSO in the doses of 1,000, 2,000, or 4,000 mg/kg bw/day (groups assigned as: BDE-209 1000 , BDE-209 2000 , and BDE-209 4000 , respectively).The doses of BDE-209 were chosen based on literature data 18,19 .Bws, water and food intake were recorded weekly.Clinical signs of poisoning were observed each day of the experiment.After the period of exposure, animals were sacrificed, and samples of blood and liver were taken.Liver weight and observed morphological changes were recorded immediately.Liver enzyme activity was measured in serum samples.Samples of the liver were taken for histopathological analysis as well as for homogenisation, and these samples were stored at -80°C prior to the analysis of the oxidative stress parameters.The rest of the liver had been stored at -20°C before the analysis of BDE-209 concentration could be performed.

Determination of BDE-209 in liver
The method was based on multistep extraction from homogenised liver, extract cleaning and determination of BDE-209 by gas chromatography (GC) -electron capture detection (GC -ECD) and GC -mass spectrometry (MS).Homogenised sample of 0.5-2 g was dried and fragmented into fine powder and transferred in a glass centrifuge tube.A total of 8 mL of mixture n-hexane:dichlormethane [(8:2; (V/V)] was added to the powder, and the total amount was vortexed, sonificated and centrifuged.Supernatant was separated, dried in air flow and than resolved in 5 mL of acetonytrile.Further cleaning of sample was done using QuEChERSs (Quick Easy Cheap Effective Rugged Safe) (ENVIRO-CLEAN r EUMIV50CT, amchro GmbH, Hattersheim, Germany).After shaking, the content was centrifuged and aliquot was transfered into the tube for solvent evaporation.The dried residue was reconstituted in 1 mL of nhexane and injected in GC.The retention time (Rt) and mass spectra (m/z values) were used for determination, and the pik area was used for quantification.Quality control of the analytical method was performed using certified reference material CIL-EDF-2524 Clean Fish (slurry) -Organic contaminants (LGC standards).

Determination of liver enzymes activities
Activity of AST, ALT, γ GT, and ALP in serum samples was determined on automatic analyser using commercial tests (Roche Elecsys 2010-cobas c 111 analyser, Roche Diagnostics, Mannheim, Germany).

Histopathological analysis of liver tissue
The liver sample was excised and fixed with 10% neutral formaldehyde.Dehydratation of tissue samples was done with graded aethanol and embedded in paraffin blocks.For semiquantitative tissue analysis, sections in 2 μm thick paraffin were stained by hematoxylin and eosin (HE) method and analyzed (Olympus-2 microscope).
Following criteria were used for semiquantitative evaluation of histopathological changes: 0 -unchanged liver`s parenchyma; 1 -single cells with intracellular oedema, vasodilatation of blood vessels and the appearance of inflammatory response cell; 2 -groups of cells with cytoplasmic vacuolation, distinct vasodilatation of numerous blood vessels (over 50%) and the accumulation of cellular infiltrate in the surrounding tissue; 3 -most cells with pro-minent vascular degeneration and karyopyknosis, focal accumulation of cellular infiltrate, 4 -vacuolar degeneration and karyolysis in all cells, single cell necrosis and diffuse accumulation of cell infiltrate, and 5 -massive and diffuse necrosis, foam cells and tissue degeneration.For determine these parameters, whole longitudinal and cross-sectional tissue was analysed and observed under high magnification (40).

Determination of oxidative stress parameters
Oxidative stress parameters were measured after homogenisation of liver samples in saccharose medium.Following parametres were determined: concentration of MDA, activity of SOD and concentration of -SH groups.Proteins were measured in homogentes by Bradford 20 method.
The level of MDA in liver homogenates was based on reaction of MDA with thiobarbituric acid under acidic condition for 15 min at 95°C in termostatic water bath.In this reaction light yellow to pink colour complexes were formed depending on the concentration of MDA in tissue homogenates.Intensity of colour was measured at 523 nm and 600 nm.
Activity of SOD, EC 1.15.1.1.in homogenates was measured using the method of Misra and Fridovich 21 , based on ability of SOD to inhibit spontaneous autooxidation of adrenaline under alkaline condition (pH 10.2).
The total content of -SH groups in liver homogenates was determined by the Ellman's 22 method which is based on the reaction of 2,2-dinitro-5,5-dithio-benzoic acid (DTNB) with alifatic thiol compounds in alkaline condition (pH 9.0).

Statistical analysis
A significance of the difference among the data in the groups for certain effect was determined by analysis of variance (ANOVA) and post-hoc Tukey test (p < 0.05) (Statistica 7.0).
PROAST software was used (RIVM, Bilthoven, Netherland) for the determination of dose-response relationship and calculation of benchmark dose lower confidence limit [(BMDL i.e.BMDL 5 if 5% of change in effect was considered as critical effect size -(CES)].Moreover, associated benchmark dose to critical dose was assigned as critical effect dose (CED) [23][24][25] .
Correlation analysis was used to estimate the relationship between external and internal dose (p < 0.05).The following equation was derived: internal dose [mg BDE-209/kg of liver wet weight (ww) = 0.0002  external dose (mg/kg bw/day) + 0.0622

In life toxicology
During the study period no clinical signs of poisoning were observed.Water consumption during the period of exposure decreased after the first week (Figure 1) and at the end of study was significantly lower in rats treated with medium and the highest dose of BDE-209 (Table 1).
During the experiment there were no significant changes in food intake related to DMSO group (Figure 1).Animals body weights were increasing during the period of exposure, but statistically significant differences in weight gain were not observed (Figure 2).
After the application of 1,000, 2,000 or 4,000 mg BDE-209/kg/day, the concentrations of BDE-209, measured in the liver, were 0.269, 0.569 and 0.859 mg/kg of the liver ww, respectively.In the control and the DMSO group concentrati-ons of BDE-209 were below the quantification limit.
The lowest concentration of BDE-209 in the liver of Wistar rats, resulted in a significant increase in relative liver weight comparing to the control, while two highest concentrations, resulted in a significant decrease in relative liver weight compared to the results recorded in the DMSO group (Table 1).Relative liver weight was uniformly changing in the dose dependant manner, and calculated BMD 5 for internal dose of BDE-209 was 0.224 mg/kg of liver ww, corresponding to external dose of 971.4 mg BDE-209/kg/day (Table 1).

Liver enzymes
As for the results of liver enzyme activity, a significant increase of the AST was induced by the medium and the highest concentrations, while a significant increase of γ-GT was induces by the lowest and the highest liver BDE-209 concentrations (Table 2).
The dose-response relationship was confirmed only for these two effects on liver enzymes.For the effect on AST, calculated BMDL 5 was 0.07228 mg BDE-209/kg of liver ww and   the curve representing the target tissue dose-response relationship is shown in Figure 3.The dose of 0.07228 mg BDE-209/kg of liver ww, corresponding to external dose of 39 mg/kg/day, was the lowest BMDL 5 among all the calculated benchmark doses indicating the effect on AST as the most sensitive biomarker of BDE-209 hepatotoxicity in subacutely exposed rats.

Histopathology
Tissue sections of the rat liver from the control and the DMSO group did not show any deviation from the normal histological structure (Figure 4, A and B).
With the lowest concentration of 0.269 mg/kg, normal histology of the liver was preserved only in the central parts.Edema of hepatocytes, mild hyperemia and small focal he-morrhages in the sinusoids were observed.Sinusoids were slightly narrowed because the moderate edema of the most hepatocytes.Cytoplasm was very eosinophilic and filled with small vacuoles.Nucleus in these cells were rounded, irregular and hyperchromatic, and nucleoli were difficult to be observed (Figure 4C).Numerous pathological mitoses were present, in most hepatocytes, particularly in the group where internal dose of BDE-209 was 0.569 mg/kg of liver ww (Figure 4D).All blood vessels were dilated, with moderate to severe polymorphonuclear cell infiltration (Figure 4E).A response higher than 10% was considered as extra risk for the degree of histopathological changes and therefore BMDL 10 was calculated.The values of the degree of histological damage as ordinal type of the variable were converted in quantal type of the variable 23,24 .Dose response was confir- med for either internal or external dose of BDE-209 and corresponding BMDL 10 doses were 0.324 mg BDE-209/kg of liver ww and 812 mg/kg bw/day, respectively.

Oxidative stress parameters
A significant influence of BDE-209 on oxidative stress parameters, MDA, SOD, -SH, in liver homogenates was not confirmed, however internal dose-response relationship was confirmed as a linear model assigned as E1 according to PROAST software (Table 3).

Discussion
Determining the relation between external and internal doses, and measurements of BDE-209 concentrations in the liver show that the ratio among the external doses (1 : 2 : 4) is different from the ratio among internal doses (1 : 2 : 3.5), implying the lower absorption when the highest dose is applied.Internal doses correlate with external ones (r = 0.972; p < 0.05) according to the equation: internal dose (mg BDE-209/kg of liver ww) = 0.0002  external dose (mg/kg bw/day) + 0.0622.Hepatotoxicity is demonstrated based on significant increase in AST and γ-GT activities, while the lowest BMDL 5 is calculated for the serum AST activity.The value of 0.07228 mg BDE-209/kg of liver ww, corresponding to external dose of 39 mg/kg/day, indicates the increase of AST activity as the most sensitive biomarker of BDE-209 hepatotoxicity in subacutely exposed rats.The degree of histological damage increases in the dose-dependent manner, and the corresponding BMDL 10 dose is 0.324 mg BDE-209/kg of the liver.
For decaBDE the United States Environmental Protection Agency provides no observed adverse effect level (NO-AEL) dose of 2.22 mg/kg bw/day as a reference point, however this dose is related with neurobehavioral effects [26][27][28] .As for the liver, in a 2-year long NTP study (1986), male and female B6C3F1 mice, administered decaBDE in the diet at the doses of 0,3200 or 6,650 mg/kg bw/day for males and 0,3760 or 7,780 mg/kg bw/day for females, the following ef-fects were recorded: liver granulomas and liver hypertrophy in males.Based on these results no NOAEL for liver effects is derived.However, the study identifies a NOAEL for a portal-of-entry effect for females of 3760 mg/kg bw/day 29 .The data sets from another NTP (1986) 2-year rat and mouse studies are selected for BMD modeling: thrombosis in the liver, liver degeneration, in male rats and centrilobular hypertrophy in livers of male mice.The lowest calculated BMDL 10 was 406 mg/kg bw/day for liver degeneration effect in male rats 29 .Only in one study internal dose, in site, was used for the assessment of BDE-209 subacute oral toxicity in rats 30 .
In this experiment internal doses arround 0.25 mg/kg of liver ww induced slight centrilobular hypertrophy in the liver together with increased expression of hepatic CYP1A and CYP2B (BMDLs 10 0.5-0.7 mg/kg bw/day) and dose-dependent decrease in serum ALP (BMDL 10 0.6 mg/kg bw/day, corresponding to 0.222 mg/kg of liver ww).Histopathological changes seen in our experiment started from the concentration of 0.269 mg/kg of liver ww, which is very close to the value published by Van der Ven et al. 30 .Hepatotoxicity of BDE-209 either in vivo or in vitro is usually connected with: a) the increased formation of reactive oxygen species, oxidative stress and decreased efficacy of antioxidative defence system 7,[31][32][33][34] ; induction of apoptosis related with oxidative stress in human hepatoma cells HepG2 33 ; aryl hydrocarbon receptor (AhR) binding particularly expression of luciferase reporter gene mediated by AhR in H4II hepatoma cell line 5 ; induction of CYP1A1 mRNA and CYP1A1 protein mediated by AhR 5,30 .The lack of effect on oxidative stress parameters in our experiment can be understood from the differences, since the values were measured at the end of exposure in which activation of adaptive mechanisms of enzyme or nonenzyme antioxidative defense could take place 35 .

Conclusion
The results of the present work add up to the issue of decabrominated diphenyl ether toxicity profile with a focus on the relationship between internal dose and hepatotoxicity.Critical internal dose for the effect on AST of 0.07 mg/kg of liver ww, corresponding to external dose of 39 mg/kg/day in our study is the lowest dose ever observed in the studies on decabrominated diphenyl ether hepatotoxicity indicating the increase of AST activity as the most sensitive biomarker of decabrominated diphenyl ether hepatotoxicity in subacutely exposed rats.Critical effect based on internal dose in the majority of cases is considered as more precisely defined than the effect established based on external dose, particularly for the persistent sub-stances with low absorption rate, such as decabrominated diphenyl ether.

Aknowledgements
The results are the part of the Project III 46009, Ministry of Education, Science and Technological Development of the Republic of Serbia.