Sustained expression of CYPs and DNA adduct accumulation with continuous exposure to PCB126 and PCB153 through a new delivery method: Polymeric implants

Abstract

A new delivery method via polymeric implants was used for continuous exposure to PCBs. Female Sprague-Dawley rats received subcutaneous polymeric implants containing PCB126 (0.15% load), PCB153 (5% load), or both, for up to 45 days and release kinetics and tissue distribution were measured. PCB153 tissue levels on day 15 were readily detected in lung, liver, mammary and serum, with highest levels in the mammary tissue. PCB126 was detected only in liver and mammary tissues. However, a completely different pharmacokinetics was observed on co-exposure of PCB153 and PCB126, with a 1.8-fold higher levels of PCB153 in the liver whereas a 1.7-fold lower levels in the mammary tissue. PCB126 and PCB153 caused an increase in expression of key PCB-inducible enzymes, CYP 1A1/2 and 2B1/2, respectively. Serum and liver activities of the antioxidant enzymes, PON1 and PON3, and AhR transcription were also significantly increased by PCB126. ³² P-Postlabeling for polar and lipophilic DNA-adducts showed significant quantitative differences: PCB126 increased 8-oxodG, an oxidative DNA lesion, in liver and lung tissues. Adduct levels in the liver remained upregulated up to 45 days, while some lung DNA adducts declined. This is the first demonstration that continuous low-dose exposure to PCBs via implants can produce sustained tissue levels leading to the accumulation of DNA-adducts in target tissue and induction of indicator enzymes. Collectively, these data demonstrate that this exposure model is a promising tool for long-term exposure studies.

Keywords: 32P-Postlabeling; CYPs; DNA adducts; PCB126 (3,3’,4,4’,5-pentachlorobiphenyl); PCB153 (2,2’4,4’,5,5’-hexachlorobiphenyl); Polymeric implants; paraoxonase 1 (PON1); polychlorinated biphenyls (PCBs).

Fig. 1

Release of PCB126 and PCB153 from polymeric implants in vitro and in vivo. (A) Structures and polymeric implants of PCB126 and PCB153. (B) Daily and cumulative release of PCB153 from polymeric implants in vitro. (C and D) Cumulative release of PCB126 (C) and PCB153 (D) from polymeric implants (one 1.5 cm × 2.6 mm dia; 0.15 and 5% loads, respectively) grafted subcutaneously into female S/D rats. Animals were euthanized after 6, 15 and 45 d; recovered implants were solvent extracted to measure the residual PCBs spectrophotometrically against a standard curve; data represent an average of three implants ± SD. Average daily dose over 45 d: 0.98 and 48.6 μg of PCB126 and PCB153, respectively. Release at 15 and 45 d was compared with 6 d release. **p < 0.01, ***p < 0.001.

Fig. 2

³²P-postlabeling/TLC analysis of liver and lung polar and lipophilic DNA adducts. (A) representative ³²P-postlabeling DNA adduct maps of lung tissue on day 15 from female S/D rats after continuous exposure to PCB126 or sham from subcutaneous polymeric implants. (B and C) DNA adducts levels in indicated tissues following 15 d of sham or PCB exposure; average daily dose over 15 d: 2.18 and 105.5 μg of PCB126 and PCB153, respectively. Data represent an average of four rats ± SD. *p < 0.05.

Fig. 3

Levels of thiobarbituric acid reactive substances (TBARS). Liver and serum of female S/D rats were analyzed following treatment with continuous low doses of PCB126, PCB153 or their combination via subcutaneous polymeric implants for 6, 15 and 45 d. A(i), B(i) and sham group in A(ii), B(ii) show the data from the 15 d time point. Average daily dose of PCB126 and PCB153 are given in Fig. 1, Fig. 2 legends.

Fig. 4

Activity of paraoxonase1 (PON1) in liver (A) and serum (B) after PCB exposure. PON1 activities in tissues of rats treated with PCB126, PCB153 and their combination for different times was determined with two substrates, paraoxon and phenylacetate. A(i), B(i) and the sham group in A(ii), B(ii) show the data from the 15 d time point. Data represented as mean ± SD (n = 4).

Fig. 5

Change in mRNA levels of PON1 (A), PON2 (B), PON3 (C), AhR (D), and APOA1 (E) in the liver of rats exposed to PCBs via polymeric implants. mRNA levels were determined by qRT-PCR and adjusted based on the level of the housekeeping gene rRPL13a. The sham group shows the data from the 15 d time point. Data represent means of four rats ± SD.

Fig. 6

Change in gene expression and protein levels of cytochrome P450s in rat livers after exposure to PCB126 and PCB153 via polymeric implants. (A and B) total RNA isolated from the liver was analyzed by qRT-PCR. Data represent average of four rats ± SD. (C) protein levels of CYP1A1, 1A2 and 1B1 in the rat liver after 15 d of treatment with indicated PCBs. Co-treatments show the time response at 6, 15, and 45 d (A and B only). The sham group shows the data from the 15 d time point. *p < 0.05, ***p < 0.001.