Evaluation of Octamethylcyclotetrasiloxane (D₄) as an Inducer of Rat Hepatic Microsomal Cytochrome P450, UDP-Glucuronosyltransferase, and Epoxide Hydrolase: A 28-Day Inhalation Study

Abstract

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Inhalation Toxicology of Octamethylcyclotetrasiloxane (D4) Following a 3-Month Nose-Only Exposure in Fischer 344 Rats

“…This observation also has been demonstrated for the classical P450 2B1/2 inducer, phenobarbital, and has led to the classi cation of D 4 as a "phenobarbital-like" inducer of hepatic microsomal enzymes in rats. The reversibility of liver weight increases observed in this study and in a previous study (Klykken et al 1999) and the results of the P450 analyses of McKim et al (1998) suggest that this effect on the liver is an adaptive response essentially identical to that elicited by classical phenobarbital-type enzyme inducers.…”

“…Increases in serum levels of°-GT have been found following exposure to ethyl alcohol (Goldberg and Martin 1975), acetominophen, barbiturates, and phenytoin (Young, Pestaner, and Gibberman 1975) in the absence of any hepatic disorder.°-GT has been found to be markedly elevated in rats following phenobarbital exposure (Remandet et al 1984). Importantly,°-GT is a microsomal enzyme and has been observed to be increased in response to microsomal enzyme induction (Suber 1989), and event known to occur following exposure of rats to D 4 , D 5 , and phenobarbital (McKim et al 1996(McKim et al , 1998. There were no histological alterations in the liver (or kidney) that supported the increases in serum levels of either°-GT or ALT.…”

Letter re: "Cyclosiloxanes produce fatal liver and lung damage in mice".

“…This is in marked contrast to all of the available literature. Studies by McKim et al (10) clearly show that CS-D4 induces cytochrome P450 2B1/B2 in rats in a time, dose-dependent, and "phenobarbital-like" manner. In other words, it is an adaptive effect.…”

Response from Lieberman and Colleagues

“…He is accurate that we do not cite any of the references he has provided in our discussion. We were in error in not including the contribution of McKim et al (12) We believe that much more work on the low molecular weight cyclosiloxanes is necessary. Inhalation studies like the one referred to by Meeks, ingestion studies like the one summarized by Lukasiak et al, and injection studies are all necessary to develop an understanding of the biologic importance of these agents.…”