Ronald A. Lubet

A pleiotropic response to phenobarbital-type enzyme inducers in the F344/NCr rat: Effects of chemicals of varied structure

The effects of a number of phenobarbital-type inducers on selected drug-metabolizing enzymes in male F344/NCr rats were determined by measuring specific catalytic activities and/or by measuring the levels of RNA which hybridize with specific probes for the corresponding genes. The effects on hepatic CYP2B1 were assessed by measuring the levels of CYP2B1-specific RNA and benzyloxyresorufin O-dealkylase and testosterone 16 beta-hydroxylase activities. Levels of CYP3A were monitored by measuring the rate of hydroxylation of testosterone at the 6 beta-position. Microsomal epoxide hydrolase activity was determined by measurement of cellular RNA specific for this form and by assaying the hydrolysis of benzo[a]pyrene-4,5-oxide. UDP-glucuronyltransferase activity was assayed by measuring the glucuronidation of 3-hydroxybenz[a]anthracene. Levels of glutathione S-transferase Ya/Yc were measured by quantifying total cellular RNA coding for the proteins. When male F344/NCr rats were administered various doses of phenobarbital or dichlorodiphenyltrichloroethane (DDT), strong correlations between the induction of CYP2B1 and the induction of epoxide hydrolase or UDP-glucuronyltransferase activities were observed. Treatment of rats with barbiturates, hydantoins, halogenated pesticides such as DDT or alpha-hexachlorocyclohexane, 2,4,5,2,4,5-hexachlorobiphenyl, CYP2B1 inhibitors such as clotrimazole or clonazepam, or such structurally-diverse compounds as 2-hexanone or diallyl sulfide resulted in induction of CYP2B1-mediated enzyme activity and induction of certain other forms of cytochrome P450, microsomal epoxide hydrolase, at least one form of UDP-glucuronyltransferase, and multiple forms of glutathione S-transferase. This suggests that, as a class, compounds which induce CYP2B1 also induce a coordinate hepatic pleiotropic response which includes induction of these other phase I and phase II drug-metabolizing enzymes.

Regulation of gene expression of various phase I and phase II drug-metabolizing enzymes by tamoxifen in rat liver.

The objective of the present investigation was to evaluate the effect of tamoxifen (TAM) on the gene expression of different phase I and phase II drug-metabolizing enzymes. Groups of male and female F344/NCr rats were administered either corn oil or TAM (2.8 to 45 mg/kg body wt x 14 days) dissolved in corn oil by gavage. An additional group of rats received a diet supplemented with phenobarbital (PB, 500 ppm). Northern blot analyses of total liver RNA were conducted using [32P]-labeled cDNA or oligonucleotide probes coding for different sulfotransferase (ST); UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), epoxide hydrolase (EPH) or cytochrome P450 (CYP) mRNA transcripts. In male rats, TAM increased the levels of STel, STa and STpl mRNAs, whereas PB increased only the STel mRNA. In female rats, there was no expression of STel and STHA mRNA in either control or TAM-treated animals. TAM and PB increased UGTBe/p mRNAs in all rats, whereas UGTml mRNA was elevated only in PB-treated animals. EPH mRNA was elevated markedly in all rats treated with TAM and PB, whereas GSTya/ye mRNA was highly increased by PB, but only marginally increased by TAM. Finally, TAM increased CYP3A1 mRNA, and slightly increased CYP2B1 mRNA, whereas PB highly elevated mRNAs for both of these CYP genes. In conclusion, treatments of rats with TAM increased the mRNA levels of many phase I and phase II drug-metabolizing enzymes, and this pleiotypic response to TAM seems to be different from other prototype inducers such as PB or dioxin (TCDD).

The chemopreventive agent diallyl sulfide: A structurally atypical phenobarbital-type inducer

Diallyl sulfide (DAS), a known chemopreventive agent, was administered i.g. (200 or 500 mg/kg body wt/day) to male F344/NCr rats for 4 days. Livers were removed, and hepatic levels of a variety of drug-metabolizing enzymes were determined with either catalytic assays or by quantifying levels of total cellular RNA coding for the individual genes of interest. The high dose of DAS induced the cytochrome P450 (CYP) 2B subfamily to near maximal levels [i.e. similar to those induced by phenobarbital (PB)] and induced the CYP3A subfamily, while having minimal effects on the levels of the CYP1A subfamily. In addition, DAS induced the glutathione S-transferase alpha subfamily, the glutathione S-transferase mu subfamily, and epoxide hydrolase. Unlike PB, however, DAS was also able to induce quinone oxidoreductase. In fact, the pleiotropic hepatic response to DAS appeared to be similar to that elicited by PB, with the exception that only DAS induced quinone oxidoreductase. Finally, we determined that DAS induced the levels of a specific nuclear binding protein that appears to be associated with the induction of various genes that are part of the pleiotropic response caused by PB-type inducers.

A mouse model for tumor progression of lung cancer in ras and p53 transgenic mice.

Although ras and p53 are the most commonly found oncogene and tumor suppressor gene, respectively, in human cancers, their collective roles in tumor progression have yet to be defined in animal models. Here, we demonstrated the synergistic effect between ras and p53 in promoting tumor progression during lung tumorigenesis using bitransgenic mice. Mice with a heterozygous knockout of K-ras (K-raswt/ko) were mated to p53 transgenic mice (p53val135/wt) in lung tumorigenesis (K-raswt/ko × p53val135/wt). F1 mice exhibited a significant increase in lung tumor load (tumor multiplicity × tumor volume) when compared to those seen in either K-raswt/ko mice or p53val135/wt mice alone. Furthermore, over 50% of the lung tumors were lung adenocarcinomas in bitransgenic mice compared to only 3% in wild-type mice. Alterations of ras and p53 appear to promote the development of lung adenocarcinomas. These results provide the in vivo experimental evidence of synergistic interactions of ras and p53 in lung tumor progression.

DETECTION OF DIFFERENTIALLY EXPRESSED GENES IN MOUSE LUNG ADENOCARCINOMAS

Increasing evidence suggests that altered gene expression is associated with the induction and maintenance of malignancy in various organs including mouse lung adenocarcinomas. A competitivecDNA libraryscreening (CCLS)was used to examinegene expression in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung adenocarcinomas from (C3H/HeJ A/J)F1 mice. Comparisons of RNA expression in lung adenocarcinomas to those of normal surrounding lung tissue revealed altered expression in 220 clones from more than 50,000 clones screened. Fifty clones were selected for quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis to verify altered expression. PCR primers were designed based on partial sequence analysis of the clones. Twenty-two clones were found to be differentially expressed in lung adenocarcinomas compared with normal lungs. GenBank database analysis showed that 14 of the 22 clones were homologous with known genes, whereas 8 clones contained novel sequences. Thirteen clones were down regulated in tumors compared to normal lung tissues, and 9 were overexpressed. The clones underexpressed or absent include adipocyte p27, carbonic anhydrase III, carbonyl reductase, cytochrome CYP2E1, skelemin, myosin, major urinary protein, and contrapsin. Overexpressed clones include Brutons tyrosine kinase, cyclin D3, poly(A)-binding protein, alpha-fetoprotein, transferrin, and mouse B2 family repetitive sequence. Further examination of biologic implications of the differentially expressed genes in lung adenocarcinomas is necessaryto understand their role(s)in mouse lung carcinogenesis.Increasing evidence suggests that altered gene expression is associated with the induction and maintenance of malignancy in various organs including mouse lung adenocarcinomas. A competitive cDNA library screening (CCLS) was used to examine gene expression in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung adenocarcinomas from (C3H/HeJ x A/J])F1 mice. Comparisons of RNA expression in lung adenocarcinomas to those of normal surrounding lung tissue revealed altered expression in 220 clones from more than 50,000 clones screened. Fifty clones were selected for quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis to verify altered expression. PCR primers were designed based on partial sequence analysis of the clones. Twenty-two clones were found to be differentially expressed in lung adenocarcinomas compared with normal lungs. GenBank database analysis showed that 14 of the 22 clones were homologous with known genes, whereas 8 clones contained novel sequences. Thirteen clones were down regulated in tumors compared to normal lung tissues, and 9 were overexpressed. The clones underexpressed or absent include adipocyte p27, carbonic anhydrase III, carbonyl reductase, cytochrome CYP2E1, skelemin, myosin, major urinary protein, and contrapsin. Overexpressed clones include Brutons tyrosine kinase, cyclin D3, poly(A)-binding protein, alpha-fetoprotein, transferrin, and mouse B2 family repetitive sequence. Further examination of biologic implications of the differentially expressed genes in lung adenocarcinomas is necessary to understand their role(s) in mouse lung carcinogenesis.

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.

A markedly diminished pleiotropic response to phenobarbital and structurally-related xenobiotics in Zucker rats in comparison with F344/NCr or DA rats

Phenobarbital (PB) and certain structurally-related compounds induce a variety of hepatic drug-metabolizing enzymes in many strains of rats. Thus, following administration of PB (300, 500 ppm), barbital (BB, 1500 ppm) or 5-ethyl-5-phenylhydantoin (EPH, 500 ppm), CYP2B1-mediated benzyloxyresorufin O-dealkylase activity and epoxide hydrolase activity were profoundly induced in female DA and F344/NCr rats. In contrast, outbred female lean and obese Zucker rats showed markedly reduced CYP2B1 responses (less than 15% and less than 5% of those observed in the female DA or F344/NCr rat) to PB (doses less than or equal to 300 ppm), BB (1500 ppm) or EPH (500 ppm). In parallel studies, profound increases in RNA levels coding for CYP2B1, glutathione S-transferases Ya/Yc (alpha subclass), or epoxide hydrolase were detected in the female F344/NCr rat following treatment with PB (300 ppm), BB (1500 ppm) or EPH (500 ppm). In contrast, lean Zucker rats showed a strong response only to the highest dose of PB (500 ppm), implying that the diminished response in the Zucker rats may occur at some pretranslational level. Similar studies with lower doses of PB, EPH or BB in male lean Zucker rats showed a decreased response, relative to that in male F344/NCr rats. However, this insensitivity was not as profound as that observed in the female Zucker rats. In fact, the response to PB-type inducers in male or female Zucker rats is probably most clearly explained as a shift of the dose-response curve sharply to the right (decreased responsiveness, compared to F344/NCr or DA rats of the same sex). This decreased responsiveness of female lean Zucker rats to induction of CYP2B1, relative to that of F344/NCr rats, was also observed with the structurally-diverse PB-type inducers clonazepam, clotrimazole and 2-hexanone. In contrast, the female Zucker rat (obese or lean) displayed a pronounced response to induction of CYP1A-mediated ethoxyresorufin O-deethylase activity by beta-naphthoflavone, a prototype inducer of CYP1A1 and CYP1A2. The Zucker rat would thus appear to represent a potentially exploitable genetic model for examining the mechanism of enzyme induction by the myriad xenobiotics which induce a PB-type response.

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.

A gene expression signature predicts chemopreventive response of an EGFR inhibitor in a rat mammary cancer model.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 AbstractsnnAbstract #1111 nnRats treated with the carcinogen methylnitrosourea (MNU) develop multiple, hormonally dependent mammary tumors. The efficacy of the epidermal growth factor receptor (EGFR) inhibitor Gefitinib (Iressa) to prevent MNU-induced mammary cancers in female Sprague-Dawley rats was determined. Rats were given a single dose of MNU (75 mg/kg body weight) at 50 days of age. Rats bearing MNU-induced small palpable cancers showed variable responses to treatment with Iressa; with approximately 50% having a complete regression and the remaining 50% having a partial or no response. We conducted exon array studies of differentially expressed genes and/or alternative splicing exons related to the responses (complete vs partial or no response). Preliminary studies showed that, although there were genes whose expression was similarly modulated in both groups, a gene expression signature can be identified that is highly predictive of which rat mammary tumors are susceptible to treatment with Iressa. Cross validation results indicated that gene expression signatures may be useful in predicting the response of a rat mammary tumor to treatment with Iressa. Currently, we are conducting validation studies in a separate set of rat mammary tumors.nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1111.

Identification of urinary biomarkers to distinguish tumor bearing and control rats in the methylnitrosourea (MNU) – induced model of mammary carcinogenesis: use of label-free, comparative, ultra-high resolution nano-LC mass spectrometry.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 AbstractsnnAbstract #1109 nnIn the MNU induced model of mammary cancer, rats given a single dose of MNU (75 mg/kg BW), via the jugular vein, at 50 days of age develop multiple ER+ mammary cancers. These cancers appear similar to highly differentiated ER+ human breast cancers, and have been shown to be sensitive to many of the same chemotherapeutic agents that are effective in women. When a female rat developed its first palpable cancer, it was placed in a metabolic cage and urine collected overnight on dry ice. The urine from these rats were compared to those obtained from control rats. Using a new sample preparation protocol, peptides were prepared from the urines of control (n = 4 individual animals) and rats bearing induced mammary tumors (n = 4). The peptide pools were analyzed using nano-LC-LTQ-FT-MS. The 8 LC-MS analyses were aligned and the integrated accurate mass signals were quantified using Rosetta ElucidatorTM software. The ion currents from 4430 peptides (charge state > +2) were analyzed between the two groups using ANOVA. Two hundred and twenty nine peptides were found to be significantly different (P< 0.01) between the two groups, with clear distinction between the urines from control and experimental animals being observed using hierarchical clustering and principal component analysis. The protein identifications that were significantly increased in the tumor-bearing urines were confirmed by targeted tandem mass spectrometry. Further studies relating the altered expression of these peptides to differences in tumor size, the effects of therapeutic agents, etc will also be presented.nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1109.