3.1 In vivo metabolism of TCC in rats
First, we obtained the full scan mass spectra of a TCC standard. The molecular ion and the most abundant fragment ions were 313 ([M-H]−), 160 ([C6H4NCl]−), and 126 ([C6H3NCl2]−). Under the experimental conditions described above, TCC eluted at 19.4 min. Because the metabolites of TCC could potentially fragment like the parent compound, we obtained a precursor ion mass spectrum of m/z=160, the most abundant fragment ion of TCC, from the rat urine samples (U1 and U2) after deconjugation and on- line SPE-HPLC separation. We observed three major precursors: A (m/z = 313, retention time [RT] = 19.4 min), B (m/z = 345, RT = 18.3 min), and C (m/z = 329, RT = 17.9 min), but only in the urine (U1 and U2) of the dosed rats (). We did not observe these three compounds in any of the three control rats urine samples or in the urine collected before dosing (U0) from the 9 dosed rats (data not shown).
Total ion chromatograms of precursor ion scans (m/z 160, 142, and 168) from urine samples collected from nine rats after the administration of TCC.
Oxidized metabolites of TCC, such as 2’-OH-TCC, had been reported to be the major biliary metabolites of TCC (Jeffcoat et al. 1977
). Therefore, we also obtained the precursor ion scans of the most abundant fragment ions for 2’-OH-TCC (m/z
= 168) and 3’-OH-TCC (m/z
= 142) from urine collected from both dosed and control rats. We found a total of six precursors of m/z
142 and m/z
168 only in the urine collected from dosed rats after administering TCC (). These precursors were B, C, D (m/z
= 329, RT=19.2 min), E (m/z
=345, RT=15.6 min), F (m/z
-295, RT=16.1 min), and G (m/z
-295, RT=16.6 min) ().
To identify these metabolites, we obtained the enhanced product ion (EPI) mass spectra of the after-dose urine (U1) after on-line SPE-HPLC separation. By comparing with the EPI data and retention times of the authentic standards, we unequivocally identified A as the parent compound TCC (), D as the oxidized metabolite 2’- OH-TCC (), and C as 3’-OH-TCC, another oxidized metabolite (). Metabolites B and E shared the same molecular ion at m/z 345, but metabolite E eluted earlier (15.6 min) than metabolite B (18.3 min). EPI scans of these two metabolites showed fragments at m/z 329, 160, 168 for metabolite B, and m/z 329, 142, 168 for metabolite E (data not shown). These data suggested that metabolites B and E were dihydroxylated-TCC isomers, with the second hydroxyl group on different sites of the ring. Similarly, we tentatively identified the two other minor metabolites F and G (both with the same molecular ion m/z 295) as a pair of dechlorinated hydroxy-TCC isomers. However, because of the lack of the authentic standards, the structures of these two pairs of isomers could not be unequivocally determined, and additional NMR experiments are needed to fully characterize the chemical structures of these compounds.
Enhanced product ion (EPI) mass spectra of different precursors in urine collected from rats after the administration of TCC: A) precursor A, B) precursor D, and C) precursor C.
We measured the total (free plus conjugated) and free concentrations of TCC and its two metabolites, 2’-OH-TCC and 3’-OH-TCC, in rats urine and serum with multiple-reaction-monitoring (MRM) after the target analytes were extracted and separated by the on-line SPE-HPLC system. To increase the accuracy of the measurements, we added D7-TCC to the samples as the internal standard for all of the three analytes. The ion transitions used for quantitation (or confirmation) and the retention time of each target analyte are listed in . shows the total and free median concentrations of TCC, 2’-OH-TCC, and 3’-OH-TCC from urine and serum samples collected from the 9 rats dosed with TCC on day 2 (pre-dose, U0), day 3 (U1), and day 4 (U2, S). We did not detect TCC or its metabolites in the pre-dose urine (U0) samples (). Similarly, we did not detect TCC or TCC metabolites in any of the urine (U0, U1, and U2) and serum samples collected from three control rats (data not shown). Within 24 hrs after dosing, TCC was rapidly metabolized to the oxidative metabolites. 3’-OH-TCC appeared to be the main metabolite, with median total urinary concentrations of 3210 ng/mL vs 102 ng/mL (2’-OH-TCC) and 16 ng/mL (TCC) (). The median urinary concentrations of 3’-OH-TCC after dosing dropped about 15% from day 3 to day 4 to 2720 ng/mL, and urinary median concentration of 2’-OH-TCC increased from 102 ng/mL to 900 ng/mL (). We also detected 2’-OH-TCC and 3’-OH-TCC in serum, but at much lower levels than in the urine collected in the same after administration; median serum concentrations were 55.1 ng/mL (2’-OH-TCC) and 33.4 ng/mL (3’-OH-TCC). All of these findings suggest that TCC metabolized rapidly in rats through phase I metabolism, and the major urinary metabolites of TCC were 3’-OH-TCC and 2’-OH-TCC.
Analyte retention time (RT), and precursor ion → product ion transitions monitored for quantitation (and confirmation)a.
Figure 3 Median total and free concentrations of TCC (A), 2’-OH-TCC (B), and 3’-OH-TCC (C) from urine (U0, U1, and U2) and serum samples (S) collected from 9 rats dosed with 500 mg/kg body weight TCC. The error bars indicate the standard deviation (more ...)
Furthermore, we detected 3’-OH-TCC mainly in its conjugated form; mean conjugate percent (%), defined as the ratio of concentrations of conjugated and total species, was 88% for urine collected in day 3 and 71% for urine collected in day 4. However, we detected TCC and 2’-OH-TCC in urine primarily in their free form, and the mean conjugate ranged from 14% to 25%. In serum, the main 3’-OH-TCC species was also its conjugate, with the mean conjugate of 3’-OH-TCC at 95%, while the mean conjugate of TCC and 2’-OH-TCC in serum were 1% and 65%, respectively.
Taken together, our data suggest that in the rat, after oral ingestion, TCC undergoes phase I metabolism to form 3’- and 2’-OH-TCC, similar to di(2-ethylhexyl) phthalate in humans (Koch et al. 2004
; Koch et al. 2005
). We speculate that most of 3’-OH-TCC was further metabolized through phase II metabolism to its conjugate before urinary excretion. By contrast, the formation and elimination of 2’-OH-TCC was slower than that of 3’-OH-TCC and did not appear to involve extensive phase II metabolism. Unfortunately, we collected urine samples for only 48 hrs after dosing the animals. Therefore, we could not determine whether additional amounts of 2’-OH-TCC conjugate would have excreted in urine more than 2 days after dosing. Nonetheless, the study design was adequate to demonstrate that TCC was rapidly metabolized through phase I and phase II metabolism, and that the oxidative metabolites 3’-OH-TCC and 2’-OH-TCC were the major urinary metabolites of TCC. Previous studies in rats (Hiles 1977
; Howes and Black 1976
; Warren et al. 1978
), monkeys, and humans (Birch et al. 1978
; Hiles et al. 1978
; Hiles and Birch 1978
; Scharpf et al. 1975
) suggest similar metabolic pathways of TCC regardless of administration route (e.g., oral, intravenous, dermal). Therefore, our results can be extrapolated to exposure routes other than oral ingestion. However, additional research is needed to identify unequivocally the metabolic profile of TCC by these other routes of exposure.
Of interest, our findings were also in agreement with a previous study which suggested that 2’-OH-TCC and 3’-OH-TCC were the main urinary oxidative metabolites based on the percentage of the extracted radioactivity after repeated oral administration of TCC to three rats (Warren et al. 1978
). However, in that particular study, the majority of the urinary radioactivity was recovered from the parent TCC, which did not happen in our study. We speculate that these differences might be related to the much higher dose used in that study (2000 ppm TCC in the diet for 10 days), which could potentially saturate the regular metabolic pathway. Furthermore, our results are also in agreement with those from a recent in vitro study suggesting that di-hydroxylated TCC and dechlorinated hydroxyl-TCC could be formed by the oxidative metabolism of TCC (Baumann et al. 2010
). However, we could not confirm the formation of quinone imines in vivo, even though these compounds were also suggested as potential oxidative metabolites of TCC in vitro (Baumann et al. 2010
3.2 Measurements of TCC, 2’-OH-TCC and 3’-OH-TCC in human urine and serum
To check the usefulness of 2’-OH-TCC and 3’-OH-TCC, the major urinary metabolites of TCC in rats, as biomarkers of human exposure to TCC for biomonitoring purposes, we measured the total and free concentrations of TCC, 2’-OH-TCC and 3’-OH-TCC in 50 urine samples collected anonymously in 2010 from adult volunteers in Atlanta, GA with no documented occupational exposure to TCC (). We also measured the same compounds in 16 commercial available serum samples collected between 1998 and 2003 from persons with no documented occupational exposure to TCC. In urine, we detected the total species of TCC, 2’-OH-TCC and 3’-OH-TCC in up to a third of the samples; the frequency of detection of TCC, 2’-OH-TCC and 3’-OH-TCC in urine ranged from 5.4% to 28% (). However, we detected TCC, not 3’-OH-TCC, at the highest frequency and highest concentration ranges in urine. The administered dose (500 mg/kg body weight) was relatively high and may have resulted in induction or inhibition of Phase I or Phase II metabolic pathways. Nonetheless, our findings are consistent with a biotransformation study conducted using rats, monkeys, and men which also suggested possible species differences (human versus rat) in TCC metabolism (Birch et al. 1978
). Furthermore, we found that the conjugated forms of TCC, 3’-OH-TCC, 2’-OH-TCC were the main species in urine. Our findings, albeit limited to a relatively small number of samples, are in agreement with early research data in which people were orally dosed with TCC (Hiles and Birch 1978
Mean and median concentrations (ng/mL) of total and free TCC, 2’-OHTCC and 3’-OH-TCC from 50 human urine.a
We did not detect 3’-OH-TCC and 2’-OH-TCC in any of the human serum samples, but we detected TCC in about 50% of the serum examined, both in total and free forms. However, TCC was detected in serum at a much lower level than in urine, with a mean concentration at 0.45 ng/mL versus 3.85 ng/mL in urine. Like for other non- persistent compounds (e.g., phthalates, bisphenol A) to which humans are likely exposed through episodic—rather than chronic—events, the concentrations in serum are much lower than the urinary concentrations. This is the main reason why urine is the preferred biomonitoring matrix for assessing exposure to non-persistent compounds (Needham and Sexton 2000
). It is also important to note that the urine and serum results presented here are not from the same individuals and collected years appart. Furthermore, the TCC serum results from these 16 samples must be interpreted with caution because TCC can be used in a variety of consumer and personal care products, and we had no information on the procedures for collection, processing, and storage of the samples analyzed. Therefore, we could not rule out the possibility of contamination with the parent compound during the collection and processing of the samples.