![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan (K.H.); Department of Food and Nutrition, Nakamura Gakuen University, Fukuoka, Japan (N.K.); and School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan (Y.K.)
(Received September 21, 2004; accepted December 16, 2004)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionConclusionReferences |
|---|
). The ratios of formation of these metabolites are dependent on the degree of substrate chlorination (Kato et al., 1980; Haraguchi et al., 1997) as well as the metabolic capacity of the species (Safe, 1994; Koga and Yoshimura, 1996). Some OH-PCBs and MeSO2-CBs are persistent and show specific retention in the blood or tissues of laboratory and wild animals (Bergman et al., 1994a,b; Oberg et al., 2002) as well as in humans (Sandau et al., 2000; Chu et al., 2003). Phenolic PCB metabolites cause alterations in thyroid hormone metabolism (Morse et al., 1996; Brouwer et al., 1998), inhibit estrogen sulfation and binding to estrogen receptors for estrogenic or antiestrogenic effects (Connor et al., 1997; Kester et al., 2002), and give rise to potentially cytotoxic dihydroxylated or quinoid PCB metabolites (Amaro et al., 1996). On the other hand, some MeSO2-CBs induce expression of several P450 isozymes (Kato et al., 1997), reduce thyroid hormone levels in rats (Kato et al., 1999), and also show antiestrogenic effects (Letcher et al., 2002). These persistent metabolites may be responsible for the toxic effects of PCBs.
Guinea pigs are the most sensitive experimental animals to the toxicity of coplanar PCBs and polychlorinated dibenzo-p-dioxins (Kociba and Cabey, 1985), whereas hamsters are less sensitive (Olson et al., 1980; Wroblewski and Olson, 1988). These differences in sensitivity may be due to metabolic activation by the unique P450-dependent monooxygenase system and substrate specificity in these species (Koga et al., 1998). For example, in vitro studies have shown that 3,4,3',4'-tetraCB (CYP1A1) is hydroxylated by rats or hamsters but not by guinea pigs (Koga et al., 1995), whereas 2,4,5,2',4',5'-hexaCB (CYP2B18) is hydroxylated by guinea pigs but hardly by rats or hamsters (Ariyoshi et al., 1997). Therefore, the different metabolic capacities of PCBs may result in the differences in distribution profiles of metabolites in animals exposed to PCBs.
In the present study, we examined the species differences in tissue distribution of PCB residues and persistent PCB metabolites among rats, hamsters, and guinea pigs 5 days after exposure to a technical PCB mixture, Kanechlor 500. This article describes the 1) comparison of the residual PCB profiles (percentage of contribution) in the liver of these three species, 2) species differences in metabolite profiles of MeSO2-CBs in the liver and of OH-PCBs in serum, and 3) GC/MS characterization of unknown metabolites retained in the serum of guinea pigs.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
; Bergman et al., 1995). Syntheses of veratrolic PCBs will be described elsewhere. The chemical names of MeSO2-CBs and OH-PCBs were simplified on the basis of the IUPAC-derived numbering system of the parent PCBs. All solvents (n-hexane, acetone, ethanol, and dichloromethane) were of pesticide grade (Kanto Chemical Co., Tokyo, Japan). Animals. Male Wistar rats (b.wt. 190–200 g), male Hartley guinea pigs (280–290 g), and male Golden Syrian hamsters (80–90 g) were housed three or four per cage in the laboratory with free access to commercial chow and tap water. Animals received a single i.p. injection of 100 mg/kg Kanechlor 500 dissolved in corn oil. Control animals received an equivalent volume of vehicle alone. The animals were sacrificed by decapitation 5 days after injection, and the liver, lungs, and serum were removed and stored at –20°C until analysis.
Isolation of Metabolites. Tissue samples were treated as described previously (Haraguchi et al., 1998). Briefly, tissues were homogenized and extracted with acetone/n-hexane (2:1, v/v). Two internal standards, 2,3,4,5,3',4',5'-heptaCB (70 ng) and 4-methyl-3-MeSO2-5,2',3',4',5'-pentaCB (24 ng), were added to each extract, and the mixtures were applied to a gel-permeation column packed with Bio-Beads S-X3 (50 g; Bio-Rad, Hercules, CA). Dichloromethane/n-hexane (1:1) was used as a mobile phase at a flow rate of 4 ml/min. The metabolite fraction (120–200 ml) was divided into phenolic and neutral fractions by partitioning with potassium hydroxide (0.5 M in 30% ethanol) and n-hexane. The potassium hydroxide phase was acidified with hydrochloric acid (2 M, 10 ml) and extracted with n-hexane/diethyl ether (9:1), and then methylated with diazomethane for 30 min at 4°C. The neutral fraction (n-hexane layer) was further subjected to silica gel column chromatography (1 g; Wakogel S-1; Wako Pure Chemicals, Osaka, Japan) by elution with 10 ml of n-hexane for PCBs and successively with 10 ml of dichloromethane for MeSO2-CBs. Serum (0.1–1 ml) was spiked with an internal standard (2,3,4,5,3',4',5'-heptaCB), acidified with 4 volumes of 0.5 M HCl and extracted with n-hexane/diethyl ether (1:1). The phenolic PCB fraction was spiked with another internal standard (2,3,4,5,6,3',4',5'-octaCB) and methylated with diazomethane. Recoveries of 4'-hydroxy-2,3,4,5,3',5'-hexaCB added to control serum and liver samples prior to extraction were 89 to 97% (n = 4) in the phenolic fraction.
Identification and Quantification. Identification of individual PCBs and phenolic PCB metabolites was performed on a GC/MS system (electron ionization-selected ion monitoring mode; QP-5000; Shimadzu, Kyoto, Japan) with a DB-5MS capillary column (60 m x 0.25 mm i.d. and 0.25-µm film thickness; J&W Scientific, Folsom, CA). The injection temperature was 250°C. The temperature program was initiated at 100°C (2 min) by 20°C/min to 250°C, and then by 2°C/min to 290°C (20 min). Helium was used as a carrier gas at a constant flow rate of 1.0 ml/min. The transfer line and quadrupole temperatures were 250 and 150°C, respectively. The mono- and dihydroxylated PCBs were monitored at molecular ion (M+) and M+ + 2 for methoxy derivatives of tetra-, penta-, and hexachlorinated congeners.
Quantification of PCBs, OH-PCBs, and MeSO2-CBs was performed on a Shimadzu GC 14A equipped with an electron capture detector (63Ni-ECD) and a split-splitless injector operated in the splitless mode. Nitrogen was used as a carrier and make-up gas. Column conditions were analogous to those described above for GC/MS. Individual PCBs and metabolites were quantified from the relative peak area to internal standards by comparison with authentic standards.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
|
MeSO2-CBs in the Liver. Figure 1 shows the ECD gas chromatograms of the MeSO2 fraction of liver extracts of the three species treated with Kanechlor 500. GC/MS analysis showed that most of the MeSO2-CBs detected were originated from 2,5-di- or 2,3,6-trichloro-substituted congeners. The levels and meta/para substitution ratios of MeSO2-CB congeners in the liver are shown in Table 2. Rats showed preferential production of meta- and para-substituted MeSO2 metabolites of 2,5,3',4'-tetraCB, 2,3,4,2',5'-pentaCB, 2,4,5,2',5'-pentaCB, 2,3,6,3',4'-pentaCB, 2,3,4,2',3',6'-hexaCB, and 2,3,6,2',4',5'-hexaCB, with a similar 3-/4-substitution ratio. Hamsters produced minute amounts of meta- and para-substituted MeSO2-CBs derived from 2,5,3',4'-tetraCB and 2,4,5,2',5'-pentaCB. In contrast, guinea pigs showed selective production of meta-substituted MeSO2-CBs derived from 2,3,6,4'-tetraCB, 2,3,6,2',5'-pentaCB, 2,4,5,2',5'-pentaCB, 2,3,4,2',3',6'-hexaCB, 2,3,4,5,2',5'-hexaCB, and 2,3,6,2',4',5'-hexaCB, although 2,3,4,2',5'-pentaCB yielded 3'- and 4'-MeSO2-2,3,4,2',5'-pentaCBs at a similar concentration ratio (Fig. 1). Total levels of MeSO2-CBs were higher in the order guinea pigs > rats > hamsters. The concentration ratios of meta- and para-MeSO2-CBs were the highest (10.1) for guinea pigs, whereas the ratios were 0.87 and 1.10 for rats and hamsters, respectively. On the other hand, marked and selective retention of 4'-MeSO2-2,4,5,2',5'-pentaCB was observed in the lungs of rats (4'-/3'-MeSO2 substitution ratio = 11), although no selective retention of para-MeSO2-CBs was observed in the lungs of guinea pigs or hamsters (data not shown).
|
|
OH-PCBs in Serum. Figure 2 shows the retention profiles of phenolic PCBs in serum of the three species after exposure to Kanechlor 500. The concentrations of major OH-PCBs and the metabolite/parent CB ratios are shown in Table 3. Rat serum showed specific retention of 4-OH-2,3,5,3',4'-pentaCB (89% contribution), most likely derived from 2,3,4,3',4'-pentaCB and/or 2,4,5,3',4'-pentaCB. On the other hand, guinea pig serum showed specific retention of 3-OH-2,4,5,2',4'-pentaCB (56%) and 3-OH-2,4,5,3',4'-pentaCB (20%). In contrast, hamster serum retained primarily 3',4'-(OH)2-2,4,5,2',5'-pentaCB, 4,5-(OH)2-2,3,6,3',4'-pentaCB, and 4,5-(OH)2-2,3,6,2',4',5'-hexaCB, as well as 3- or 4-hydroxy metabolites derived from 2,4,5,2',4'-pentaCB and 2,4,5,3',4'-pentaCB (Fig. 2). Thus, phenolic PCB levels showed the following order: hamsters > rats > guinea pigs. The ratios of 3-OH-2,4,5,2',4'-pentaCB/2,4,5,2',4'-pentaCB were higher in hamsters (12.3) and guinea pigs (12.0) than in rats (0.3). The ratio of (4-OH-2,3,5,3',4'-pentaCB + 3-OH-2,4,5,3',4'-pentaCB)/(2,3,4,3',4'-pentaCB + 2,4,5,3',4'-pentaCB) was higher in rats (6.3) than in guinea pigs (0.6).
|
|
Metabolites in the Serum of Guinea Pigs. The phenolic fraction in the serum of guinea pigs contained several persistent metabolites with different molecular weights from OH-PCBs or MeSO2-CBs, although no such metabolites were detected in rats and were formed at lower levels in hamsters. Figure 3 shows the GC/MS total ion chromatogram of phenolic fraction (Ma–Md, at longer GC retention times than MeSO2-CBs) in the serum of guinea pigs after exposure to Kanechlor 500. The electron impact mass spectra of Ma to Md are shown in Fig. 4. By GC/ECD and GC/MS analyses, Ma was tentatively identified as methoxy-MeSO2-tetraCB (M+, m/z = 398), and Md as a methoxy-MeSO2-pentaCB (M+, m/z = 432) due to a fragment ion, [M – 81]+ (due to loss of oxygen and OCH3Cl from M+), and [M – 144]+ (loss of OCH3Cl-O-SOCH2 from M+). Mb showed a weak molecular ion (m/z = 400) and an abundant fragment at [M – 32]+ (loss of two oxygen atoms from M+), which was characteristic of vicinal methoxy-methylthio-pentaCB. Mc showed a weak molecular ion (m/z = 416), and fragment ion [M – 16]+ (loss of oxygen) as a base peak that was characteristic of vicinal methoxy-methylsulfinyl pentaCBs. Thus, the PCB metabolites in the serum of guinea pigs were tentatively identified as hydroxy-MeSO2-tetraCB for Ma, hydroxy-methylthio-pentaCB for Mb, hydroxy-methylsulfinylpentaCB for Mc, and hydroxy-MeSO2-pentaCB for Md.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
; Kaminsky et al., 1981), there has been only limited comparative analysis of in vivo metabolism of PCBs between hamsters and guinea pigs (Koga and Yoshimura, 1996). The results of the present study indicated that the tissue distribution profiles of PCB residues and OH- and MeSO2-CBs were largely dependent on the chlorination patterns classified into four groups. This may have been due to differences in substrate specificity of each species, especially between guinea pigs and hamsters, following administration of Kanechlor 500.
PCB Residues in the Liver. The percentage composition of residual PCBs in the liver indicated that guinea pigs had lower elimination ratios of coplanar PCBs with 4-, 3,4-, or 3,4,5-chlorine substitution (group A in Table 1) than rats or hamsters. For example, the composition of 3,3',4,4'-tetraCB, which originally accounted for 0.03% in Kanechlor 500, increased to 1.8% in the livers of guinea pigs 5 days after exposure, whereas the values were 0.02 and 0.06% in the livers of rats and hamsters, respectively (Table 1). These observations suggest that guinea pigs have lower ability to metabolize coplanar PCBs. This was supported by the in vitro observation that 3,3',4,4'-tetraCB can hardly be metabolized by liver microsomes from MC or phenobarbital-treated guinea pigs, whereas it is metabolized effectively by those of rats and hamsters treated with MC (Koga et al., 1995). The reduced elimination of coplanar PCBs may partially explain the higher sensitivity to the toxicity of coplanar PCBs in guinea pigs.
On the other hand, hamsters exhibited reduced elimination of the 2,4,5-trichloro-substituted CB congeners (group C) compared with rats or guinea pigs. This observation suggests that hamsters have a reduced capability to hydroxylate 2,4,5-trichloro-substituted CBs than the other two species. The results of a previous in vitro study indicated that 2,4,5,2',4',5'-hexaCB can hardly be metabolized by rats or hamsters, whereas it is metabolized effectively by guinea pigs (Ariyoshi et al., 1997). This may be due to the lower levels of induction of CYP2B1/2 in rats and hamsters compared with guinea pigs. CYP3A may also be related to the metabolism of these PCBs, depending on species. Furthermore, it is noted that nonpersistent CBs with 2,5- or 2,3,6-chlorine substitution (group D) tend to be eliminated more slowly in hamsters than in rats or guinea pigs. The reduced elimination of these congeners is probably due to the lower catalytic activity of CYP2B in hamsters than in rats (Ishida et al., 1991; Koga et al., 1995), or to the lower abilities of phase II isozymes, such as glutathione S-transferase, to conjugate for subsequent excretion in hamsters.
MeSO2-CBs in Liver. The MeSO2 metabolites detected in the present study were consistent with the results of previous studies regarding metabolism of PCB congeners with 2,5- or 2,3,6-chlorine substitution (Haraguchi et al., 1997, 1999). However, the levels and metabolite profiles in guinea pigs and hamsters were quite different from those in rats. Hamsters formed minute amounts of meta- and para-MeSO2-CBs from 2,5,3',4'-tetraCB and 2,4,5,2',5'-pentaCB, whereas guinea pigs produced much higher levels of MeSO2-CBs and showed selective distribution as meta-substituted-MeSO2-CBs derived from CBs with 2,3,6-chlorine substitution. These results indicate that the meta-position of the 2,3,6-trichlo-substituted phenyl ring in the molecule is the preferred site for methylsulfonylation and guinea pigs may have substrate-specific phase II isozymes that catalyze the nucleophilic reaction of arene oxide intermediates with sulfhydryl groups. Indeed, the selective retention of meta-MeSO2-CBs has been observed in the livers of otters, minks, and seals in Canadian and Swedish water (Bergman et al., 1994b). Especially, 5'-MeSO2-2,3,4,2',3',6'-hexaCB was the most abundant in human tissues (Chu et al., 2003). Kato et al. (1997) reported that only meta-substituted MeSO2-CBs strongly induced CYP2B1/2 and reduced the serum thyroid hormone level in rats. Exposure of minks to a synthetic mixture of meta- and para-MeSO2-CBs also resulted in the selective retention of meta-MeSO2-CBs in the liver and increased induction of some hepatic CYP2B isozymes as well as alteration of thyroid hormone levels in blood (Lund et al., 1999). Higher levels of induction of CYP2B1/2 by MeSO2-CBs as well as induction of CYP3A-like activity (Schuetz et al., 1986, 1998) may alter the metabolic pathways of individual PCBs in rats and contribute to the high level of toxicity of nonplanar PCBs.
Phenolic PCBs in Serum. For the three species examined in the present study, persistent OH-PCBs in serum were derived metabolically from 2,4,5-trichloro-substituted CBs (e.g., 2,4,5,2',4'-pentaCB, 2,4,5,3',4'-pentaCB, 2,3,4,2',4',5'-hexaCB, and 2,4,5,2',4',5'-hexaCB) via direct insertion of a hydroxyl group into the 3-position or via epoxidation and subsequent NIH chlorine shift at the 4-position. Indeed, all the OH-PCBs detected in the present study have been observed in gray seal and in human blood (Bergman et al., 1994a; Sandau et al., 2000); however, the levels of OH-PCBs and metabolite/parent CB ratios were species-specific since different species have different types of P450 enzymes (Koga et al., 1998).
Exposure of animals to Kanechlor 500 resulted in higher levels of elimination of 2,4,5,3',4'-pentaCB in rats than in guinea pigs or hamsters. For this metabolism, rats showed selective formation of 4-OH-2,3,5,3',4'-pentaCB (metabolite/parent CB ratio = 6.3), whereas guinea pigs showed selective formation of 3-OH-2,4,5,3',4'-pentaCB (ratio = 0.59). Hamsters showed formation of 3-OH-2,4,5,3',4'-pentaCB and 4-OH-2,3,5,3',4'-pentaCB in a ratio of 1:3 (ratio = 3.0). Although serum does not necessarily have the same profile as liver, the differences in the levels of OH-PCBs and bio-transformation ratios in serum may be explained by the species-dependent metabolic capacities. In fact, these results are consistent with the in vitro observation that 2,4,5,3',4'-pentaCB can be metabolized readily to 4-OH-2,3,5,3',4'-pentaCB (CYP1A1/2) by MC-inducible hepatic microsomes of rats, whereas it shows a lesser degree of metabolism to 2- or 3-OH-2,4,5,3',4'-pentaCB (CYP2B18) by guinea pig liver microsomal P450 isozymes (Koga et al., 2002).
For metabolism of 2,4,5,2',4'-pentaCB, both guinea pigs and hamsters showed selective formation of 3-OH-2,4,5,2',4'-pentaCB (transformation ratios = 12.0 and 12.3, respectively), whereas this PCB was relatively resistant to metabolism by rats (ratio = 0.27). On the other hand, the elimination of 2,4,5,2',4',5'-hexaCB and 2,3,4,2',4',5'-hexaCB was delayed in rats and hamsters, resulting in lower distribution of their metabolites in the liver. These findings were supported by those of previous in vitro studies indicating that 2,3,4,2',4',5'-hexaCB and 2,4,5,2',4',5'-hexaCB can be hydroxylated effectively by the guinea pig cytochrome P450 isoform CYP2B18 but are resistant to metabolism by rats or hamsters (Koga et al., 2001).
Other persistent phenolic PCBs were catechol metabolites derived from CBs with 2,5- or 2,3,6-chlorine substitution (e.g., 2,4,5,2',5'-pentaCB, 2,3,6,3',4'-pentaCB, and 2,3,6,2',4',5'-hexaCB), which were retained in the serum of hamsters at concentrations similar to those of monohydroxylated PCBs. The formation of catechol may involve a two-step direct hydroxylation process or a pathway via arene oxide and dihydrodiol intermediates. Thus, the tissue distribution ratios of catechols in hamsters and of MeSO2-CBs in guinea pigs would reflect the extreme differences in metabolism of CBs with 2,5- or 2,3,6-chlorine substitution. Although their mechanisms of retention are still unknown, catechols may act as endocrine disruptors, similar to OH-PCBs, with affinity to the thyroid hormone transport protein transthyretin, concomitant with a reduction in circulating thyroid hormone level (Brouwer et al., 1998) or to estrogen receptor with estrogenicity (Garner et al., 1999).
The phenolic PCB fraction in the serum of Kanechlor 500-treated guinea pigs contained several additional metabolites that were observed in GC/MS total ion chromatograms at longer GC retention times than those of MeSO2-CBs (Fig. 3). Two of these, Ma and Md, were identified tentatively as hydroxy-MeSO2-tetraCB and -hexaCBs, respectively. The other two, Mb and Mc, were presumably the precursors of Md because when the fraction was oxidized chemically, the two peaks were converted to the corresponding methylsulfones (Md). Mio and Sumino (1985) identified 3-hydroxy-4-methylthio-2,5,2',5'-tetraCB in the feces from rats, mice, and guinea pigs dosed with 2,5,2',5'-tetraCB, suggesting that the precursor of the metabolite was 3,4-epoxide. The epoxide may have been opened by nucleophile attack of the sulfhydryl group and subsequently converted to the hydroxylated metabolite by dehydrogenation (Bakke et al., 1982). Therefore, the metabolites retained in the serum of guinea pigs may be congeners containing a hydroxyl group in the position adjacent to the MeSO2 group.
|
Conclusion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: PCB, polychlorinated biphenyl; P450, cytochrome P450; OH, hydroxyl; CB, chlorobiphenyl; OH-PCB, hydroxylated PCB; MeSO2-CB, methylsulfonyl PCB; GC/MS, gas chromatography-mass spectrometry; GC/ECD, gas chromatography-electron capture detection; MC, 3-methylcholanthrene.
Address correspondence to: Dr. Koichi Haraguchi, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan. E-mail: ko-ichi{at}ka2.so-net.ne.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. Kato, S.-i. Ikushiro, R. Takiguchi, K. Haraguchi, N. Koga, S. Uchida, T. Sakaki, S. Yamada, J. Kanno, and M. Degawa A Novel Mechanism for Polychlorinated Biphenyl-Induced Decrease in Serum Thyroxine Level in Rats Drug Metab. Dispos., October 1, 2007; 35(10): 1949 - 1955. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
