The Distribution of Metabolites of 2,2',3,4,4',5',6- Heptachlorobiphenyl (cb183) in Rats and Guinea Pigs

The in vivo metabolism of 2,2',3,4,4',5',6-heptachlorobiphenyl (CB183) was studied in rats and guinea pigs. Similarly to the in vitro metabolism, 3'-hydroxy (OH)-CB183 and 5-OH-CB183, were found as major metabolites in the serum and the feces of both species (serum < feces). Phenobarbital (PB)-pretreatment of animals increased the production of both metabolites, whereas 3-methylcholanthrene (MC)-pretreatment suppressed the CB183 metabolism in rats and guinea pigs. Our previous study reported that 5-methoxy (MeO)-CB183 had the same retention time as 4-MeO-2,2',3,4',5,5',6-heptachlorobiphenyl (CB187) on DB-1 capillary column. However, the use of SP-2330 capillary column resulted in an excellent separation of 5-MeO-CB183 and 4-MeO-CB187. From these results, it is concluded that 4-OH-CB187 could not be produced from CB183 in rats and guinea pigs. Introduction 4-OH-metabolites of PCB congeners have been detected in human blood at higher concentrations and have been shown to possess various toxicological activities to disturb homeostasis of thyroid hormone and vitamin A in animal blood, to behave as an estrogen or antiestrogen, to inhibit estrogen sulfotransferase, to decrease cell-cell communication in gap junction and also to act as an agonist for thyroid hormone receptor. Among them, 4-OH-CB187 which is a PCB metabolite with the highest concentration in human blood, is thought to be presumably from CB187 or CB183, a minor component in PCB preparations. Recently, we have demonstrated by the in vitro and in vivo studies that 4-OH-CB187 could be produced from CB187 in rats and guinea pigs and was exclusively detected in the blood but not in the feces. 10 Moreover, we have reported that CB183 was metabolized to two OH-metabolites by liver microsomes of rats and guinea pigs. From the data of GC-MS, one metabolite (M-1) was determined to be 3'-OH-CB183 but the methylated derivative of another one (M-2) looked like 4-MeO-CB187 or 5-MeO-CB183 in terms of retention time in GC. Therefore, to clarify whether 4-OH-CB187 is formed from CB183 or not, we examined the in vivo metabolism of CB183 in rats and guinea pigs. We report here that 4-OH-CB187 is not a metabolite of CB183 in both species. Materials and Methods CB183, 4-MeO-CB187 and 5-MeO-CB183 were synthesized by the method of Cadogan. 3'-OH-CB183 was synthesized by the method of Hutzinger et al. and methylated by diazomethane. Twelve male Wistar rats (body wt. about 200 g) and twelve male Hartley guinea pigs (body wt. about 300 g) were divided into untreated, PBand MC-treated groups and administered PB and MC ip at a dose of 80 and 20 mg/kg/day for two days, respectively. Two days after the last injection of PB and MC, CB183 was injected ip at a single dose of 80 μmol/kg. Animals were sacrificed 4 days after administration of CB183 and blood was isolated. The feces were pooled during the experiment. Dry powdered feces were extracted with acetone-n-hexane (2:1, v/v) for 24 h in a Soxhlet apparatus. The serum (0.5 ml) was acidified with 0.5 M sulfuric acid (0.25 ml) and then extracted with chloroform-methanol (2:1, v/v) and n-hexane. The extracts were methylated with diazomethane and applied to GC-ECD (HP5890 Series II). CB183 and its metabolites TOXICOLOGY II (CANCER AND OTHER CHRONIC EFFECTS) Organohalogen Compounds Vol 69 (2007) P-173 1761 were quantified by a calibration curve of authentic CB183 for GC peak area. The GC conditions were as follows: column, DB-1 (30 m x 0.25 mm, 0.25 μm thickness) or SP-2330 (30 m x 0.25 mm, 0.25 μm thickness) capillary column; carrier gas, N2 (1 ml/min); column temp., 230oC; injection port temp., 250oC; detector temp., 250oC. Results and Discussion Previously, we reported that 4-OH-CB187 can be produced from CB183 in the in vitro study using animal liver microsomes because the retention times of 4-MeO-CB187 and a CB183 metabolite (M-2), completely agreed in DB-1 capillary column (30 m length). However, when SP-2330 capillary column (30 m length) was used, a candidate of CB183 metabolite (5-MeO-CB183) and 4-MeO-CB187 could be separated with the retention times of 23.69 min and 24.13 min, respectively (Fig. 1). As a result of reexamination of all metabolites found in the in vitro study and in this study, no 4-OH-CB187 was found in CB183 metabolism (data not shown). Table 1 shows the concentrations of unchanged CB183 and two metabolites, 3'-OH-CB183 (M-1) and 5-OH-CB183 (M-2), in the serum of rats and guinea pigs 4 days after exposure to CB183. In untreated rats, in addition to CB183 (6.29 nmol/ml of serum), M-1 and M-2 were detected at the concentrations of 0.23 and 0.37 nmol/ml of serum, respectively. In untreated guinea pigs, the concentrations of CB183, M-1 and M-2 were 4.74, 1.00 and 0.17 nmol/ml of serum. M-1 was about 6 times higher level than M-2. Similarly to our in vitro study, 11 PB-treatment increased both M-1 and M-2 to 1.5to 1.7-fold of untreated in rats and only M-2 to 1.8-fold of untreated in guinea pigs. On the other hand, MC-treatment decreased both metabolites to less than 60 % of untreated ones in both species. These results suggest that PB-treatment is more effective to accelerate CB183 metabolism in rats and guinea pigs. Fecal excretion of CB183, M-1 and M-2 in rats and guinea pigs are shown in Table 2. In addition to unchanged CB183, both M-1 and M-2 were observed in 4-days feces at much higher concentration than those in the serum of both animal species. M-1 and M-2 were 5.7 and 3.4 nmol/g of dry feces in untreated rats and also 4.1 and 0.8 nmol/g of dry feces in untreated guinea pigs, respectively. PB-treatment accelerated fecal excretions of M-1 and M-2 to about 2 to 3-fold of untreated rats and increased them slightly in guinea pigs. MC-treatment decreased both metabolites in rat feces and M-2 in guinea pig feces in the similar manner to those in the serum. Our previous study showed that, in guinea pigs dosed with CB187, 4-OH-CB187 was distributed exclusively to the blood but was not found in the feces at all. In this study, we observed relatively high amount of M-2 (5-MeO-CB183) in the feces of guinea pigs and rats. Thus, the result that M-2 showed a different distribution pattern from 4-OH-CB187 supported the fact that M-2 is not 4-OH-CB187. The postulated pathways in rats and guinea pigs are shown in Fig. 2. We found two metabolites (M-1 and M-2) in the serum and feces of rats and guinea pigs and the chemical structures of M-1 and M-2 were finally determined to 3'-OH-CB183 and 5-OH-CB183, respectively. It is apparent that both metabolites are produced by a direct hydroxylation mechanism by PB-inducible cytochrome P450 isoforms such as rat CYP2B1 and guinea pig CYP2B18. 11 Fig. 1 Separation of two synthetic standards, 4-MeO-CB187 and 5-MeO-CB183, by GC-ECD with SP-2330 capillary column 0 10 20 30 Retention time (min) Cl Cl Cl