Lisa E. Beebe

Induction of hepatic CYP1A activity as a biomarker for environmental exposure to Aroclor® 1254 in feral rodents

Specimens of the feral mouse species Reithrodontomys fulvescens trapped from a polychlorinated biphenyl (PCB)-contaminated field location had hepatic ethoxy-resorufin (ETR) O-dealkylase activities and immunoreactive CYP1A protein contents which were two- to threefold higher than those measured in animals of the same species and sex collected from non PCB-contaminated reference sites. Specimens with hepatic ETR O-dealkylase activities differing by as little as 50% could readily be assigned as originating from the PCB or reference sites by the use of a specific chemical inhibitor of cytochrome P450IA (CYP1A). The relative levels of ETR O-dealkylase activity in R. fulvescens significantly correlated with hepatic PCB burdens (r=0.819, P<0.01). When the magnitudes of the induced ETR O-dealkylase activities corresponding to given hepatic PCB burdens were compared between the feral animals, F344/NCr rats (Rattus norvegicus) or B6C3F1 mice (Mus musculus) exposed in the laboratory to dietary Aroclor® 1254, the order of sensitivity to the inducing effects of PCBs were F344/NCr rat>B6C3F1 mouse>R. fulvescens.

Mechanism-based inhibition of mouse P4502b-10 by selected arylalkynes

Suicide inhibitors of cytochrome P450 families are excellent tools to predict which isoforms mediate the metabolism/activation of a variety of chemical agents. We compared the inhibitory effects of several arylalkynes on mouse cytochromes P450 with published data for the rat model. The inhibition of P4502b specific dealkylation of benzyloxyresorufin by 2-ethynylnaphthalene (2-EN), 5-phenyl-1-pentyne (PPY), 4-phenyl-1-butyne (PBY), and 9-ethynylphenanthrene (9-EPh) was measured in hepatic microsomes from male mice treated with 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) to induce cytochrome P4502b. Pulmonary microsomes were prepared from untreated mice. 9-EPh, 2-EN, and PPY caused a time-, concentration-, and NADPH-dependent loss in P4502b activity in both tissues. PBY, however, demonstrated this type of inhibition only in liver microsomes. The IC50 was calculated for both liver and lung microsomes and compared with published Ki (concentration required for half-maximal inhibition) or KI (concentration required for half-maximal inactivation) values for the rat. PPY, PBY, and 9-EPh were equally effective inhibitors of mouse P4502b and rat P4502B1. 2-EN was a 5- to 10-fold less potent inhibitor of mouse P4502b, as compared with the rat, even though it was shown to bind to the active site of the mouse isoform as demonstrated by its metabolism to 2-naphthylacetic acid. These data suggest that the active site of the mouse P4502b enzyme is functionally similar to the rat P4502B isoform, with the exception of the disparity in its susceptibility to inactivation by 2-EN as measured by the Ki values.

Induction of hepatic CYP1A in male F344/NCr rats by dietary exposure to Aroclor 1254: Examination of immunochemical, RNA, catalytic, and pharmacokinetic endpoints

Male F344/NCr rats were exposed to low dietary concentrations of Aroclor 1254 (0-33 ppm) for 7 days, following which the induction of selected hepatic drug metabolizing enzymes was monitored. CYP1A1, measured indirectly by assaying the O-dealkylation of ethoxyresorufin in 9000 g supernatants, was increased 1.5-, 3-, 8-, and 37-fold following 7 days of exposure to 1.0, 3.3, 10, and 33 ppm Aroclor, respectively. In contrast, the O-dealkylation of benzyloxyresorufin, an indirect measure of CYP2B1 activity, was increased approximately 4-fold following exposure to 33 ppm dietary Aroclor. Measurement of the non-P450-mediated activities epoxide hydrolase, DT-diaphorase, and aldehyde dehydrogenase (NADP+, benzaldehyde) revealed < 4-fold inductions following feeding of 33 ppm Aroclor. In view of the relatively high sensitivity of the CYP1A-specific catalytic endpoint as a biomarker for Aroclor exposure, alternative endpoints for detecting induction of this subfamily of P450 were also examined. The extent of in vivo CYP1A induction was assessed by measuring serum concentrations of zoxazolamine 150 min following an intraperitoneal dose of 100 mg/kg body wt. Slight decreases in serum zoxazolamine concentration were observed in rats exposed to as little as 1.0 ppm dietary Aroclor 1254, while profound decreases were seen in rats exposed to > or = to 10 ppm Aroclor. Immunodetection of CYP1A1 protein, with a monoclonal antibody directed against this cytochrome, revealed a 2.9-fold increase in rats exposed to as little as 1.0 ppm Aroclor, and approximately 10- and 44-fold increases following exposure to 3.3 and 10 ppm dietary Aroclor, respectively. Increases in total hepatocellular RNA coding for CYP1A1 and CYP1A2, quantified by hybridization to specific oligonucleotide probes, corresponded well to the increases in hepatic O-dealkylase activity for ethoxyresorufin (CYP1A1) and methoxyresorufin (CYP1A2), respectively. Thus, CYP1A induction, directly or indirectly measured with a variety of endpoints, represents a highly sensitive biomarker for exposure to relatively low doses of Aroclor 1254 in the rat.

Effect of dietary Aroclor 1254 exposure on lung and kidney cytochromes P450 in female rats: evidence for P4501A2 expression in kidney

In this report, we have investigated the effect of dietary exposure to Aroclor 1254 (1-100 ppm) given chronically or discontinuously over an 84-day time interval to the female F344 rat. Cytochrome P4501A was quantified in lung and kidney by measuring the dealkylation of ethoxyresorufin substrate and by Western immunoblotting. P4501A displayed a dose- and time-dependent increase in both extrahepatic organs. The kidney appeared to be more responsive to induction than lung at all doses (maximum of 500-fold induction following 84 days exposure to 100 ppm). Further, there was evidence by enzymatic activity, immunoblotting and Northern analysis of total RNA for the presence of 1A2 in the most highly induced kidneys. The decline in 1A induction observed following discontinuous exposure was more prominent in the kidney than in the lung. These data demonstrate the sensitivity of kidney to P4501A induction capacity as compared to lung, although the persistence of the induction response was evident in lung and not kidney.