Identification of sulfated metabolites of 4-chlorobiphenyl (PCB3) in the serum and urine of male rats

Abstract

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semivolatile and have been reported in urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague-Dawley rats (150-175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 μmol/kg body weight of PCB3. Urine was collected every 4 h; rats were euthanized at 36 h; and serum was collected. LC/MS analysis of urine before and after incubation with β-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 h, with peak excretion occurring at 10-20 h after exposure. The major metabolites were 4'PCB3sulfate, 3'PCB3 sulfate, 2'PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4'PCB3 sulfate was 6.18 ± 2.16 μg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC-PCBs by analysis of phase II metabolites in urine.

Figure 1. Structure of standards used in this study

The atom numbering scheme is indicated for PCB3.

Figure 2. TIC showing metabolites of PCB3 in urine after 10 hours of exposure

(A) Five major metabolites (peaks 1–5) and eight minor metabolites (peaks a–h) were identified in the urine. The most abundant ions corresponding to the peaks were m/z 475 (a), m/z 397 (b), m/z 366 (1), m/z 366 (2), m/z 461 (c), m/z 379 (d), m/z 329 (e), m/z 348 (f), m/z 348 (g), m/z 313 (h), m/z 299 (3), m/z 283 (4), m/z 203 (5). (B) Incubation with β-glucuronidase resulted in the disappearance of peaks a, b and d, and were attributed to be sulfated-glucuronide diconjugate, dihydrodiol glucuornide conjugate, and monophenol-glucuronide conjugate respectively. (C) Incubation with both β-glucuronidase and sulfatase resulted in additional disappearance of peaks 3 and 4, which were a presumed catechol sulfate, and 4’PCB3 sulfate respectively. All monohydroxylated isomers co-eluted and are indicated by peak 5. All other remaining peaks (1, 2, c, e, f, g, and h) were not affected by hydrolyzing enzymes. MS analysis was carried out by LCQ deca. Mass spectra of all the ions are found in supplemental figure S3.

Figure 3. Effect of formic acid on the fragmentation of m/z 366 (peaks 1 and 2) to m/z 348

Extracted ion chromatograms for m/z 348 under two conditions of sample preparation in the urine sample. When no acids were used in workup procedure, four peaks 1, 2, f and g were observed (top EIC). In the urine samples diluted with equal volume of 1% formic acid (v/v), peaks 1 and 2 were degraded resulting in a single large peak corresponding to f and g (bottom EIC). A representative mass spectrum for peak 1 showing abundance of masses of ions m/z 366 and m/z 348. The spectra of peak 2 was similar to peak1. This fragmentation pattern suggested that 1, 2, f, and g are putative mercapturic acids metabolites. MS analysis was carried out by LCQ deca.

Figure 4. Free and conjugated forms of two major phenols in urine

Incubation with sulfatase released approximately six times more phenol than it was as free-phenol for both 3’-OH-PCB3 and 4’-OHPCB3. Incubation with glucuronidase did not significantly increased the mono-phenols. MS analysis was carried out by Waters Acquity TQD using an Acquity UPLC BEH C18 column, and pH of mobile phase was 10.5. (n=3)

Figure 5. Chromatograms showing PCB3 sulfate standard and their peak correspondence in urine and serum

Chromatograms of 2’ PCB3 sulfate, 3’PCB3 sulfate, 4’PCB3 sulfate, and IS in concentration of 0.05, 0.1, o.5 and 0.5 µg/mL (A), urine from PCB3 treated rats after four hours of exposure (B), urine from control animal (C), serum from PCB3 treated rats (D), and serum from control rats (E). MS analysis was carried out Waters Acquity TQD.

Figure 6. Time-course of the excretion of PCB3 sulfates in urine

Sulfates were rapidly excreted in urine, reaching a peak excretion within 10–20 h. Urine collected at each time point (50µL) was diluted 1:1 with 1% formic acid and cleaned up in SLE+ chromatography column. Analysis was carried out as described in Figure 5. (n=3)

Scheme 1. Proposed biotransformation pathway for 4-Chlorobiphenyl (PCB3) in rats

Potential enzymes/mechanisms are indicated by numbers in the legend.