Spatial Distribution of CYP2B1/2 Messenger RNA within the Rat Liver Acinus following Exposure to the Inducers Phenobarbital and Dieldrin

Abstract

Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague-Dawley-derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16beta-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment.

Global Liver Proteomics of Rats Exposed for 5 Days to Phenobarbital Identifies Changes Associated with Cancer and with CYP Metabolism

“…The present proteomic result of increased CYP2B6 (CYP2B2) agrees with earlier microarray (Seidel et al, 2006) and amplified fragment length polymorphism (Elrick et al, 2005) data. It is also supported by our earlier work in which CYP2B2 mRNA levels and P450-mediated enzyme activities were increased following the same PB treatment (Dail et al, 2007). Additional evidence in the top network for the metabolism/reactive oxygen mechanism of PB hepatocarcinogenesis is suggested by the strong increase in HIF1A which has recently become the focus of intense research regarding antioxidants and cancer.…”

“…Animal treatment and tissue processing. Adult male Sprague Dawleyderived rats were housed and fed as described previously (Dail et al, 2007). Three rats were treated ip with either saline (controls) or PB in saline at 80 mg/kg body weight/day (treated) for 5 days as described previously (Ma and Chambers, 1995).…”

“…Three rats were treated ip with either saline (controls) or PB in saline at 80 mg/kg body weight/day (treated) for 5 days as described previously (Ma and Chambers, 1995). Following sacrifice on day 5, the livers were removed and stored at À 80°C as previously described (Dail et al, 2007).…”

Current Status of Automation in the Process of Visualisation and Analysis: What is important for Toxicologic Pathology?

“…Laser microdissection has been used to investigate mechanisms of toxicity in specific compartments of the liver 15 , lung [16][17][18][19] , brain 20 , eye 21 , uterus 22 , testis 23 , and prostate 24 . It also was applied to address mechanisms of carcinogenicity in toxicity studies 25,26 .…”

The Effect ofIn VitroDieldrin Exposure on the Rat Paraoxonase 1 (Pon1) Promoter

“…Its use was discontinued in the United States in the 1970s; however, as one of the persistent organic pollutants (POPs), dieldrin still exists in the soil of rural regions throughout the world. Concentrations have been reported from about 12 ppb in Pakistan and the Mississippi Delta of the United States to 1.44 ppm in Kenya . Accumulation in exposed individuals is still a possibility since dieldrin is lipophilic and readily absorbed following inhalation, ingestion, or skin contact .…”