2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-Induced Accumulation of Biliverdin and Hepatic Peliosis in Rats

Marjo Niittynen*,1, Jouni T. Tuomisto*, Seppo Auriola{dagger}, Raimo Pohjanvirta*,{ddagger},§, Paula Syrjälä{ddagger}, Ulla Simanainen*, Matti Viluksela* and Jouko Tuomisto*

* Department of Environmental Health, Laboratory of Toxicology, National Public Health Institute, Box 95, FIN-70701 Kuopio, Finland; {dagger} Department of Pharmaceutical Chemistry, University of Kuopio, Box 1627, FIN-70211 Kuopio, Finland; {ddagger} National Food and Veterinary Research Institute, Kuopio Department, Box 92, FIN-70701 Kuopio, Finland; and § Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Box 57, FIN-00014 University of Helsinki, Helsinki, Finland

Received July 9, 2002; accepted September 16, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread, persistent, and highly toxic environmental pollutant. The most TCDD-sensitive and the most TCDD-resistant rat strains (Long-Evans [Turku/AB] and Han/Wistar [Kuopio], respectively) were crossbred to separate the alleles of two genes (Ahrand an unidentified gene "B") mediating resistance against TCDD toxicity. During crossbreeding, a new type of toxicity in livers of both sexes was detected, characterized macroscopically by intense dark green to black color and swelling that appeared most frequently after a large dose (300 µg/kg or more as a single intragastric dose) and a follow-up period of more than three weeks. Therefore, studies were undertaken to identify the causative pigment chemically and to examine the hepatotoxicity histologically. The pigment fractions were separated by thin layer chromatography and then analyzed by HPLC and electrospray mass spectrometry. The pigment was found to consist of biliverdin and several biliverdin-related compounds. In liver histopathology carried out on male rats, progressive sinusoidal distension and hepatic peliosis with membrane-bound cysts were seen. The clinical manifestations of pigment accumulation were recorded most often in intermediately resistant rat lines such as line B (homozygous for the geneB), but never occurred in rats expressing only the Han/Wistar (Kuopio)-type Ah receptor with an altered transactivation domain structure.

Key Words: TCDD; biliverdin; bilirubin; hepatotoxicity; accumulation; porphyrin metabolism; peliosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated dibenzo-p-dioxins (PCDDs) are widespread, persistent, and highly toxic environmental pollutants. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener among PCDDs and the most thoroughly investigated model compound of this class of chemicals. These compounds typically elicit a variety of biological and toxic responses ranging from induction of cytochrome P450 (CYP) isoforms 1A1, 1A2, and 1B1 to reproductive and developmental defects, immunotoxicity, thymus atrophy, endocrine imbalance, altered intermediary metabolism, liver toxicity, cancer, and wasting syndrome (reviewed by Pohjanvirta and Tuomisto, 1994Go).

Sensitivity to TCDD varies greatly among mammalian species and also among strains of the same species. The largest interstrain difference reported in rats occurs between the Han/Wistar (Kuopio; H/W) and Long-Evans (Turku/AB; L-E) substrains with LD50 values > 9600 and 10–20 µg/kg TCDD, respectively (Pohjanvirta and Tuomisto, 1994Go). Recent studies revealed that the exceptional TCDD resistance of H/W rats mainly derives from a point mutation in intron 10 of its aryl hydrocarbon receptor (AhR) gene and a consequent alteration in the transactivation domain of AhR protein (Pohjanvirta et al., 1998Go; Tuomisto et al., 1999Go). It was also previously demonstrated that there are probably two separate genes affording resistance to TCDD (Pohjanvirta, 1990Go), the other one being still unidentified and therefore called "gene B." Subsequently, classical crossbreeding methods were used to segregate these two genes from H/W and L-E parent strains into novel rat lines (Tuomisto et al., 1999Go). Three lines resulted from the breeding experiments: line A homozygous for the mutated AhR (Ahrhw/hw, hw denoting an allele originating from the H/W strain) and normal gene B (Bwt/wt, wt denoting a wild-type allele from the L-E strain); line B homozygous for the auxiliary resistance allele (Bhw/hw) and normal AhR (Ahrwt/wt); and line C homozygous for wild-type alleles of both resistance genes (Tuomisto et al., 1999Go). LD50 values for lines A, B, and C were > 10000, 400–800, and 20–40 µg/kg TCDD, respectively. Thus, line A was as resistant as H/W, line C almost as sensitive as L-E, and line B was intermediately resistant.

In necropsies carried out on TCDD-treated rats during the H/W x L-E crossing, several rats in F1, F2, and F2 x L-E generations were surprisingly discovered to exhibit a previously unseen liver toxicity syndrome. The most severe cases had large (more than double the expected size) swollen livers that were mottled and dark green or black in color. When the tissue was cut with a knife, several milliliters of dark green to black fluid leaked out. These findings prompted us to chemically and histologically characterize the responsible pigment and thus to gain further insight into the possible metabolic disturbances in these rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
TCDD (CAS# 1746-01-6; mw 321.9; purity > 99% as analyzed by gas chromatography-mass spectrometry) was purchased from UFA Oil Institute (Ufa, Russia). It was dissolved in corn oil (Sigma, St. Louis, MO). The composition of the buffer used in liver perfusions was 25 mM N-(2-hydroxyethyl)piperazine-N`-(2-ethanesulfonic acid) (HEPES)/1.5 mM ethylenediaminetetraacetic acid (EDTA)/10% glycerol/1 mM dithiothreitol, pH 7.4 (abbreviated as HEGD). HEGD reagents were purchased from Sigma. Biliverdin dihydrochloride (80% purity), hemin chloride, and Tris-Cl (Trizma, reagent grade) were obtained from Sigma. In preliminary studies standard solutions of biliverdin and hemin were made according toBonkovsky et al.(1986)Go. In mass spectrometric analysis 0.1 mM solution of biliverdin in methanol was used as a standard. Sucrose (analytical grade) was purchased from BDH Laboratory Supplies (Poole, England). Analytical grade acetone and chloroform were purchased from Labscan Ltd. (Dublin, Ireland). Concentrated hydrochloric acid and trichloroacetic acid were obtained from Merck (Darmstadt, Germany). Water was purified with a Milli-Q Water purification system (Millipore, Bedford, MA). Methanol (HPLC-grade) and formic acid (98%) were purchased from J. T. Baker (Deventer, Netherlands).

Animal husbandry.
All rats were obtained from the breeding colony of the National Public Health Institute, Kuopio, Finland. They were housed in groups in stainless-steel wire-mesh cages with pelleted R3 or R36 feed (Ewos, Södertälje, Sweden) and tap water available ad libitum. The temperature in the animal room was 21 ± 1°C, relative humidity 50 ± 10%, and lighting cycle 12/12 h light/dark. The study plans were approved by the Animal Experiment Committee of the University of Kuopio and the Kuopio Provincial Government (permits 29.1.96/5Zd, 29.1.96/8Zd, 19.6.97/47Zd, and STO90/5.2.98).

Macroscopic examinations.
A large number of rats (323) were tested with TCDD during the production, selection, and characterization of the new lines A, B, and C (for details, see Tuomisto et al., 1999Go). These rats included the three new lines, their hybrid offspring with L-E rats, and H/W x L-E F2 generation rats. They represented both genders and various age groups (4–57 weeks at the time of exposure) and were treated intragastrically with a wide range of single TCDD doses (10–2000 µg/kg). The rats were euthanized a day before the expected death or at six weeks postexposure. However, several rats were found dead although effort was made to identify moribund rats. Most nonsurvivors lived two to four weeks after treatment. All rats were examined macroscopically at necropsy.

Instrumentation.
Two different liquid chromatographic systems were used. First, we used Waters HPLC (Waters, Milford, MA) equipped with Waters autosampler 717 and Waters diode array detector 996. In liquid chromatographic separations before mass spectrometric detection the Rheos 4000 HPLC (Flux Instruments, Danderyd, Sweden) equipped with LaChrom autosampler L-7200 Merck Hitachi (Hitachi, Tokyo, Japan) and Spectroflow 757 UV detector was used. In both HPLC-systems the same Symmetry C18 column (5 µm; 4.6 x 150 mm) with a guard column (Waters) was used. Mass spectrometric analysis was performed using a Finnigan LCQ ion trap mass spectrometer fitted with an electrospray ionization source (San Jose, CA).

Pigment extraction.
The livers for the preliminary HPLC-analyses were from an experiment designed to verify the genotype of line B by determining the sensitivity of male and female hybrid offspring following line B x L-E matings. The rats were dosed intragastrically with 50 or 100 µg/kg TCDD at the age of 8–10 weeks and monitored for 42 days. They were then killed by decapitation. However, a few rats were ether-anesthetized and the livers were further perfused with HEGD to reduce the amount of blood heme in the samples.

Later, when an experiment primarily for pigment extraction was planned, line B female rats were chosen for studies because the incidence of the syndrome was high and the rats were homozygous in respect of the resistance genes (Ahrwt/wt, Bhw/hw). Rats were dosed intragastrically with 300 µg/kg TCDD at the age of 9–11 weeks. This dose was chosen because it is large enough to cause the syndrome with somewhat high incidence but it is usually not lethal to these rats. After 3 to 5 weeks, all rats were ether-anesthetized and livers were perfused with HEGD.

In preliminary studies the pigment was extracted according to Bonkovsky et al.(1986)Go with minor modifications (samples were centrifuged and filtered using 0.45 µm Spartan 30/B filter [Schleicher & Schuell, Dassel, Germany] before HPLC-analysis). Later and in mass spectrometric analysis the extraction procedure was as follows. Liver homogenates (10%, w/v) were prepared in 0.25 M sucrose/20 mM Tris-Cl (pH = 7.4) using a teflon-pestled Potter-Elvehjem glass homogenizer in a Heidolph homogenizer device. Proteins were precipitated by adding 0.8 ml 15% trichloroacetic acid to 0.5 ml of homogenate. The resultant mixture was vortex-mixed and centrifuged (10,800 x g, 4 min, room temperature). Uncolored supernatant was discarded. Precipitate was suspended in 0.8 ml of ice-cold acetone/concentrated HCl (97.5:2.5, v/v), vortex-mixed, and centrifuged (10,800 x g, 4 min, room temperature). Supernatants of 10 parallel samples were combined. The resulting solution, which now included the pigment, was concentrated under nitrogen to a final volume of about 500 µl. At this stage, the mixture appeared in two phases. The lower one contained the pigment and its volume was 50–80 µl. The upper phase was discarded. Samples were kept on ice during extraction.

Preparative thin-layer chromatography.
Concentrated pigment was separated to its components by applying it on silica gel plates (SIL G-25, Macherey-Nagel, Düren, Germany). Development was done with chloroform:methanol:water (65:25:3). Bands were scraped off the plates and suspended in methanol (300 µl). The resultant mixture was vortex-mixed and silica was removed by centrifugation. Methanol extracts were filtered using 0.45 µm Spartan 30/B filter (Schleicher & Schuell, Dassel, Germany) and stored at –20°C for further analysis.

Liquid chromatography.
In preliminary studies, liquid chromatography (solvents, gradient, and flow rate) was performed according to Bonkovsky et al.(1986)Go using UV-VIS–detection at wavelength 377 nm. Spectral data between 250 and 750 nm was collected. In mass spectrometric analysis the following liquid chromatography conditions were used. Methanol containing 0.2% formic acid and water with 0.2% formic acid were used as eluents. A 20 min gradient was employed to increase methanol from 50 to 100%. Methanol was maintained at 100% for 5 min, after which the column was restored to initial conditions in 2 min. An automatic injection volume of 20 µl with a flow-rate of 1 ml/min was used. The eluate from HPLC column was divided in a 1:9 ratio. The smaller part was directed to the mass spectrometer and the larger to the UV detector. UV detection was carried out at 377 nm.

Electrospray mass spectrometry.
All extracts were analyzed using positive ion mode. The capillary temperature was 225°C, source voltage 4 kV, sheath gas flow 100 (arbitrary units, scale 0–100 units), capillary voltage 20 V, and tube lens offset 10 V. Maximum ion time was 200 ms.

Histopathology.
In the histological examination, ten-week-old male rats of line B were used. The rats were dosed 300 µg/kg intragastrically and decapitated 2, 7, 14, 28, or 35 days later. Liver samples were preserved in 10% neutral buffered formalin, dehydrated, embedded in paraffin wax and cut to the thickness of 5 µm. The tissue slices were mounted on glass slides, stained with Mayer's hematoxylin and eosin or sudan IV, and examined using light microscope independently by two pathologists (P.S., R.P.).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Necropsy Observations
In the course of the crossbreeding to separate the resistance genes, a large number of rats of both genders and of various age groups (4–57 weeks) were treated with a wide range of TCDD doses (10–2000 µg/kg) and subjected to macroscopic examination at necropsy. When the first observations of the black liver syndrome were made, these findings were recorded from all rats during subsequent necropsies. Due to the heterogeneous rat population, all age groups were combined and two dose groups were formed, namely "low" (10–100 µg/kg) and "high" (300–2000 µg/kg).

The majority of severe black liver syndrome cases were recorded in line B, line B x L-E, or line A x L-E rats, i.e., in rats with at least one resistance allele. In these lines, the incidence of the syndrome increased with dose. In line B males the incidence was 4/27 (15%) with a low dose and 9/20 (45%) with a high dose. In line B females the incidences were 2/22 (9%) and 25/48 (52%), respectively. In line A x L-E males there were no cases (0/5) with the low dose, and the high dose resulted in 3/6 (50%) incidence. In line A x L-E females the incidences were 1/13 (8%) and 4/9 (44%), respectively.

Intriguingly, however, no cases appeared in line A rats or other rats homozygous for the resistance allele of the AhR gene (Ahrhw/hw) although 14 line A males and 22 females were treated with doses of 700–2000 µg/kg. No black livers were seen even in three line A males that were given extremely high total doses of 12,000, 18,000, and 30,000 µg/kg TCDD (unpublished data). In addition, 6 males and 9 females from H/W x L-E F2 generation with verified Ahrhw/hw phenotypes were treated with 1000 µg/kg and no black livers were seen.

Sensitive lines (C and C x L-E), which due to their sensitivity were only given low doses (10–100 µg/kg), exhibited only a few mild cases. It is not clear at this point whether this is a true line difference or whether it merely reflects the lower doses used in these lines. In line C males and females the incidences were 2/40 (5%) and 0/35, respectively. In line C x L-E males and females there were 5/9 (56%) and 0/4 cases, respectively.

In all these cases the incidences were at least that mentioned above; they may have been slightly higher, because postmortem changes in liver had made classification unreliable in a few rats that were found dead (these were classified as "no syndrome"). The proportion of these rats was highest in line C females (23%) and in the low dose group of line A x L-E males (20%), and it was 10% or less in all other groups.

Although the incidence increased with dose, the proportion of severe and mild black liver syndromes did not show a dose response according to the data from lines that were treated both with the high and the low dose. In the groups with at least 3 cases of the syndrome, the proportion of severe cases was 25–66% of all cases irrespective of the dose.

The age of rat at exposure does not seem to be a critical factor in the development of the syndrome. The syndrome was observed both in rats exposed at the age of 5 weeks as well as in rats exposed at the age of 50 weeks. Usually it took 3–5 weeks for syndrome to develop. Of all observations, only 6 out of 99 were made before day 21 postexposure.

Histopathology
In the TCDD-treated rats, a characteristic liver response (Pohjanvirta et al., 1989Go; Pohjanvirta and Tuomisto, 1994Go) was discernible from day 7 on. This consisted of swollen hepatocytes with hydropic degeneration, formation of giant hepatocytes with multiple nuclei, necrosis of single cells or of small foci, increased number of mitotic figures, vacuolization and infiltration of inflammatory cells in sinusoids. There was interindividual variation in severity of these manifestations of TCDD toxicity. Additionally, however, in the present study peculiar histopathological changes were found in line B rats that have never been recorded in L-E or H/W rats in response to TCDD or other dioxin congeners. From day 28 on, hepatic sinusoids displayed a clear tendency towards distension. This dilatation was progressive and reached a hepatic peliosis-like stage with membrane-bound cysts by day 35 in 4 out of 5 rats; one of these showed a mild black liver syndrome at necropsy. Some of the cysts appeared empty while others contained proteinaceous material, erythrocytes, or leukocytes (Figs. 1B–1DGo). At later phases, sinusoidal distension was accompanied by mild to moderate hepatic fibrosis and modest bile duct proliferation.



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FIG. 1. Histological findings in male rats of line B, 35 days after 300 µg/kg TCDD intragastrically (B–D) or corn oil treatment (A). The strikingly enlarged sinusoidal spaces in the TCDD-treated animal are shown at three magnification levels. (A) Control, magnification x40 (HE). (B) 300 µg/kg TCDD, 35 days, magnification x40 (HE). (C) The same liver as in (B), magnification x100 (HE). (D) The same liver as in (B), magnification x400 (HE).

 
One of the TCDD-treated rats of line B, which had to be euthanized on day 22 in a moribund state, exhibited excessive hepatic steatosis.

Preliminary HPLC Analyses
The dark green color of the pigment suggested that it could contain biliverdin, a heme breakdown product. We first analyzed the pigment with mere extraction followed by HPLC-analysis (Bonkovsky et al., 1986Go). However, the resulting chromatograms were not unambiguous. In the chromatograms of black liver samples there were a number of poorly separated peaks near the retention time of biliverdin. Many of these peaks had UV-VIS spectrum similar to that of the biliverdin standard. We concluded that the peak that had identical retention time and UV-VIS spectrum with the standard was likely to be biliverdin. The next task was the identification of the peaks that had similar UV-VIS spectra but different retention times. The largest peak in the chromatograms was assumed to be heme on the basis of similar UV-VIS spectrum and identical retention time with an authentic standard.

Based on these observations the procedure was further modified as follows. Livers were perfused in order to diminish the amount of blood heme. The perfusion changed the color of the livers from red with black spots to pale with green spots. This indicates that the pigment of interest was indeed green. For more efficient extraction of pigment, the extraction procedure was optimized (see Materials and Methods). In addition, the pigment was purified by preparative thin-layer chromatography prior to further analysis. These modifications improved the reliability of identification of the compounds, which was finally completed by mass spectrometry.

Thin-Layer Chromatography
The green liver pigment from a TCDD-treated rat separated into three main bands by preparative thin-layer chromatography (TLC; Fig. 2Go). The approximate Rf values were 0.67 (band 1, most polar), 0.71 (band 2), and 0.87 (band 3, least polar). Bands 1 and 2 had a bluish-green color. Band 3 was green, having some brown pigment mixed in it. In control samples from untreated rats bands 1 and 2 were missing. Instead, there was a band having about the same Rf value as band 3 in a sample from a TCDD-treated rat, but the color of this band was reddish-brown without any green pigment.



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FIG. 2. Thin-layer chromatogram of hepatic pigments from female rats of line B, 35 days after 300 µg/kg TCDD intragastrically or vehicle. BV, biliverdin standard. The three TLC bands analyzed further are numbered.

 
Identification of Compounds
Parallel to MS detection, UV detection was also carried out at 377 nm, the absorption maximum of biliverdin hydrochloride in methanol (Lemberg and Legge, 1949Go). UV detection was used to confirm that the compounds detected by MS also absorbed light at the same wavelength as biliverdin. This could be used as supporting information in the identification procedure. Only compounds absorbing light at 377 nm are considered below.

The TLC band 3 was found to mainly consist of biliverdin and heme.2 The identification of biliverdin was based on a similar retention time to an authentic standard and on the information from MS-MS tandem mass spectrometry. Both parent and daughter ion spectra of the putative biliverdin in sample were identical with the authentic standard (Figs. 3 and 4GoGo). The positive ion mass spectra of biliverdin showed an (M + H)+ ion at m/z 583.3 (calculated from atom weights: 583.7; Table 1Go). The other main component of TLC-band 3 was expected to be heme, which showed an [M(protoporphyrin IX) – 2H + Fe3+]+ ion at m/z 616.3 (calculated 616.5).



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FIG. 3. HPLC-chromatogram of biliverdin standard at 377 nm (a); corresponding base peak ion chromatogram (b) with identified compounds marked; molecular ion peak (M + H)+ 583.3 of biliverdin (c); and its fragment ion peaks (d) in positive ion mode. The structure of biliverdin and its presumed fragmentation pattern are shown (c). BV, biliverdin; BVME, biliverdin methyl ester; BVDME, biliverdin dimethyl ester.

 


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FIG. 4. The analysis of the TLC-band 3. HPLC-chromatogram at 377 nm (a); corresponding base peak ion chromatogram (b) with identified compounds marked; molecular ion peak (M + H)+ 583.3 of expected biliverdin (c); and its fragment ion peaks (d) in positive ion mode. BV, biliverdin; H, heme; BVME, biliverdin methyl ester; HME, heme methyl ester.

 

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TABLE 1 Compounds Found in Green Liver Pigment Extracted from the Livers of Line B Female Rats 35 Days after a Single TCDD Dose
 
The TLC band 1 was found to mainly consist of a compound showing an (M + H)+ ion at m/z 759.3 (Fig. 5Go). This is proposed to be biliverdin monoglucuronide on the basis of the following evidence. First, the calculated molecular mass of biliverdin monoglucuronide is 758.7, so it should show an (M + H)+ ion at m/z 759.7. Second, there was a daughter ion with the same m/z 583.2 as for biliverdin (Table 1Go). There was also a daughter ion at the m/z 297.2, which is also a daughter ion of biliverdin. Third, on the basis of Rf-value and retention time the compound with m/z 759.3 (M + H)+ is more polar than biliverdin. Glucuronidation is a typical conjugation reaction in biotransformation of toxic compounds in the liver in order to make them more soluble in water. Biliverdin accumulation in liver is an abnormal situation that could lead to the conjugation of biliverdin with glucuronate. Alternatively, biliverdin monoglucuronide might result from oxidation of bilirubin monoglucuronide, a normal intermediate in heme catabolism.



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FIG. 5. The analysis of the TLC-band 1. HPLC-chromatogram at 377 nm (a); corresponding base peak ion chromatogram (b), identified compound marked; molecular ion peak (M + H)+ 759.3 of putative biliverdin monoglucuronide (c); and its fragment ion peaks (d) in positive ion mode. The structure of biliverdin monoglucuronide and presumed fragmentation pattern are shown (c). BVMG, biliverdin monoglucuronide.

 
The main component of the TLC band 2 was a compound showing an (M + H)+ ion at m/z 741.4 (compound I in Table 1Go, Fig. 6Go). This is equivalent in size to a biliverdin monoglucuronide with a loss of a hydroxyl group. Again, the daughter ions at the m/z 583.3 and m/z 297.2 were found. Loss of hydroxyl group could also explain why this compound is slightly less polar than biliverdin monoglucuronide.



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FIG. 6. The analysis of the TLC-band 2. HPLC-chromatogram at 377 nm (a); corresponding base peak ion chromatogram (b), identified compounds marked; molecular ion peak (M + H)+ 741.4 of compound I (c); and its fragment ion peaks (d) in positive ion mode. Comp. I, compound I; BVMG, biliverdin monoglucuronide; BVMGME, biliverdin monoglucuronide methyl ester.

 
The green pigment was also found to contain compounds that showed (M+H)+ ions at m/z 597.3 and 773.4 (Table 1Go). We propose that these compounds are biliverdin methyl ester (mw = 596.7) and biliverdin monoglucuronide methyl ester (mw = 772.7), respectively (Fig. 7Go). Daughter ion patterns (Table 1Go) support this hypothesis. Because biliverdin methyl ester (and dimethyl ester) were also found in the standard solution, it seems possible that methyl esters are formed spontaneously in methanol. Furthermore, the relative amount of the putative methyl esters compared with free biliverdin remarkably increased during storage of the standard solution.



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FIG. 7. Structures and presumed fragmentation patterns of biliverdin methyl esters (a and b) and biliverdin monoglucuronide methyl esters (c and d).

 
As shown in Table 1Go, the putative biliverdin monoglucuronide methyl ester eluted at retention times of both 6.6 min and 7.7 min. Also daughter ion profiles differ at different retention times. Because biliverdin monoglucuronide has two carboxylic groups there are also two possible forms of biliverdin monoglucuronide methyl ester. Different polarities of these compounds can result in different retention times.

The areas corresponding to the TLC bands 1, 2, and 3 were also scraped off the control TLC separation (Fig. 2Go), extracted and analyzed with LC-MS. The control extract of bands 1 and 2 did not show any absorption peaks at 377 nm. The main compounds found in the band 3 control extract were identified as heme and heme methyl ester on the basis of their retention times and the information from MS-MS tandem mass spectrometry. The putative heme methyl ester showed an [M(protoporphyrin IX methyl ester) – 2H + Fe3+]+ ion at m/z 630.3 (calculated 630.5). No biliverdin or its derivatives were found in control samples.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TCDD is known to affect both the synthesis and degradation of heme giving rise to porphyria and jaundice, respectively (Pohjanvirta and Tuomisto, 1994Go; see Fig. 8Go for flowchart of heme metabolism). The porphyrinogenic effect of TCDD results in accumulation of uroporphyrin in the liver due to inhibition of uroporphyrinogen decarboxylase. CYP1A2 appears to have a critical role in the development of this uroporphyria (Smith et al., 2001Go). The mechanism of jaundice is more obscure at the moment. TCDD elevates both total and conjugated bilirubin levels in serum rapidly and progressively (Unkila et al., 1994Go). The increase in serum bilirubin could be due to diminished clearance (Choe and Yang, 1983Go; Yang et al., 1983Go) or augmented formation (Mitchell et al., 1990Go) of bilirubin in the liver by TCDD. On the other hand, in congenitally jaundiced Gunn rats a single dose of TCDD reduced serum bilirubin levels by inducing hepatic bilirubin catabolism (Cohen et al., 1986Go).



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FIG. 8. Heme biosynthetic and degradation pathways. The known effect of TCDD in heme biosynthesis is marked. ALAD, {delta}-aminolevulinate dehydratase; ALAS, {delta}-aminolevulinate synthase; BRO, bilirubin oxidase; BVR, biliverdin reductase; CPO, coproporphyrinogen oxidase; FEC, ferrochelatase; HCB, hexachlorobenzene; HO, heme oxygenase; PBGD, porphobilinogen deaminase; PCB, polychlorinated biphenyl; PPO, protoporphyrinogen oxidase; UCoS, uroporphyrinogen III cosynthase; UDP-GT, UDP-glucuronyltransferase; UROD, uroporphyrinogen decarboxylase. The flowchart (including the effect of TCDD) is based on Maines (1999)Go, Murray (1988)Go, and Sassa and Maines (2001)Go. aThe presence of bilirubin oxidase in rat liver has been demonstrated (Cardenas-Vazquez et al., 1986Go; Yokosuka and Billing, 1987Go) but the role of this enzyme in vivo is obscure.

 
The present study reveals a novel heme-related syndrome caused by TCDD. It is characterized by large black livers, which accumulate a green pigment. The syndrome mainly affected the intermediately TCDD-resistant rat lines such as line B. Both sexes were affected with about equal frequency. An interesting observation was that the syndrome was never seen—not even after massive doses of TCDD (up to 30,000 µg/kg given to a single animal)—in line A or in other rats expressing only the H/W-type AhR. This suggests a pivotal involvement of AhR transactivation domain in the molecular mechanism of the syndrome. It also implies that the syndrome belongs to type II dioxin effects, where the magnitude of effect varies broadly among different genotypes (Simanainen et al., 2002Go; Tuomisto et al., 1999Go).

Based on our current findings, the green pigment accumulating in the livers of TCDD-treated rats is likely to consist of biliverdin and its derivatives (biliverdin monoglucuronide, compound I, and methyl esters of biliverdin and biliverdin monoglucuronide). Of these, only biliverdin was identified here with the aid of a standard compound. Further analysis using standards (some of which are not readily available) for the other compounds also would verify their nature. We assume that biliverdin monoglucuronide is formed in the liver either from biliverdin or from bilirubin monoglucuronide. It is somewhat surprising that no biliverdin diglucuronide was found in the pigment. The reason for this is not known. However, it has been shown that when bilirubin conjugates exist abnormally in human serum, they are predominantly monoglucuronides (Murray, 1988Go). The putative methyl esters of biliverdin and biliverdin monoglucuronide are probably formed during sample preparation. The exact nature and formation of compound I are not known.

Biliverdin is an intermediate compound formed in heme breakdown and oxidation in the reticuloendothelial system. It is rapidly reduced to bilirubin, which is transported to liver, glucuronized, and excreted to bile (Fig. 8Go). Upstream induction of heme degradation is thus one possible explanation for increased biliverdin levels. This pathway has also other functions in addition to heme metabolism. Heme oxygenase (HO) is the only enzyme capable of producing carbon monoxide (CO), an important stress mediator and neurotransmitter. Biliverdin and iron are the by-products of CO production, which is catalyzed by two isozymes of HO, the inducible HO-1 and the constitutively expressed HO-2. Hexachlorobenzene stimulates HO-1 in rat liver (Stonard et al., 1998Go). Hexachlorobenzene and other polyhalogenated compounds are also thought to cause oxidative stress, a strong inducer of HO-1, via cellular hepatic iron and CYP1A (Maines, 1999Go; Stonard et al., 1998Go). On the other hand, biliverdin reductase displays extensive microheterogeneity in rat organs, and bromobenzene has been shown to selectively suppress the main variant in liver but not in spleen (Huang et al., 1989Go). Phosphorylation state critically regulates biliverdin reductase activity (Salim et al., 2001Go), and TCDD modifies protein kinase activities (Enan and Matsumura, 1995Go). Thus, TCDD treatment could conceivably lead to an imbalance between the hepatic activities of HO and biliverdin reductase. The present findings warrant studying whether TCDD modulates the function of these enzymes.

Another possible source of biliverdin is oxidation from bilirubin (Fig. 8Go). A number of studies have suggested during the last few years that bilirubin is an important antioxidant acting as radical scavenger in various forms of oxidative stress (Elbirt and Bonkovsky, 1999Go; Galbraith, 1999Go; Ryter and Tyrrell, 2000Go; Stocker et al., 1987Go). Oxidative stress might lead to increased oxidation of bilirubin to biliverdin, instead of glucuronidation and excretion, although experimental data are conflicting in this respect (Dudnik and Khrapova, 1998Go; Minetti et al., 1998Go).

To the best of our knowledge, the peliosis-like sinusoidal distension found here has not been reported before in the livers of TCDD-treated rats. However, it has been recorded in rats treated daily for 91 days with 3 or 10 µg/kg 2,3,7,8-TBDD, i.e., the corresponding brominated dioxin congener (Ivens et al., 1993Go). Furthermore, repeated exposure to hexachlorobenzene is capable of eliciting the lesion (Arnold et al., 1985Go; Smith et al., 1993Go), and iron overload potentiates the effect (Smith et al., 1993Go). Hepatic peliosis is frequently seen in aged Long-Evans Cinnamon rats (an animal model for copper accumulation in the liver [Wilson's disease]; Onaya et al., 2000Go). These findings suggest involvement of oxygen radicals in the pathogenesis of the condition. Another contributing factor might be the TCDD-induced wasting, since hepatic peliosis has been observed in patients with chronic wasting diseases such as tuberculosis or cancer (Burger and Marcuse, 1952Go; Hamilton and Lubitz, 1952Go). Thus, biliverdin accumulation and sinusoidal distension may share common pathogenetic factors. They further displayed similar temporal patterns. However, since only one of the four peliotic rats at 35 days exhibited the black liver syndrome, these two phenomena may be coinciding but causally unrelated.

In the present study the incidence of the black liver syndrome was very similar in both sexes, but the histopathological examination was limited only to male rats. However, in a previous study with hexachlorobenzene female rats were reported to be even more sensitive than males to hepatic peliosis (Arnold et al., 1985Go), the principal morphological finding of the present study. Therefore, it seems likely that females also would exhibit hepatic peliosis. Indeed, the macroscopic findings were the same in both sexes.

In conclusion, we have demonstrated that TCDD may, in certain conditions, cause a dramatic accumulation of bile pigments, notably biliverdin and its conjugates, and bring about peliosis-like histopathological changes in rat liver.


    ACKNOWLEDGMENTS
 
We thank Ms. Arja Tamminen and Ms. Minna Voutilainen for excellent technical assistance and Dr. Veli-Matti Kosma for work leading to the first histological observations of this syndrome. This study was supported by the Academy of Finland, the Finnish Research Program on Environmental Health (Project 42551), and the European Commission (Contracts ENV4-CT96-0336 and QLK4-1999-01446).


    NOTES
 
1 To whom correspondence should be addressed. Fax: +358 17 201265. E-mail: marjo.niittynen{at}ktl.fi. Back

2 This analysis is qualitative in nature. Therefore only rough estimates of the relative amounts of compounds are given assuming an equal response of these compounds in MS detection. Back


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