Downregulation of drug transport and metabolism in mice bearing extra-hepatic malignancies

There is increasing evidence of a systemic inflammatory response associated with malignancy, which may have an impact on both drug disposition and resistance to cytotoxic therapy. The impact of inflammation on drug disposition was studied in mice bearing a number of common tumour xenografts. C57BL/6 mice were inoculated with tumour xenografts. Hepatic expressions of Cyp3a and drug transporters were analysed at the mRNA, protein and functional levels (Cyp3a only). Circulating serum cytokines and the hepatic expression of acute phase proteins (APPs) were measured. Intratumoral levels of multidrug resistance genes were determined. Tumour xenografts elicited an inflammatory response that coincided with repression in hepatic Cyp3a11 activity and the expression of a number of hepatic drug transporters. With tumour growth, a progressive reduction in hepatic Cyp3a11 mRNA expression was seen. Conversely, an increase in the hepatic APP expression and circulating interleukin (IL)-6 levels was observed. Furthermore, a correlation was seen between increased intratumoral expression of the multidrug resistance gene, Mdr1a, and levels of circulating IL-6. Malignancy results in reduced hepatic drug disposition that correlates with an associated inflammatory response. Reduction of inflammation may improve the clinical outcome for patients receiving chemotherapeutic agents that undergo hepatic metabolism.     

P450-expression in brain tumors

Oxazaphosphorines are inactive anticancer prodrugs that are bioactivated by hepatic cytochrome P450. Besides hepatic metabolism, there is increasing interest in the possibility of intratumoral activation of oxazaphosphorines by P450. Therefore, we investigated the expression of P450 (CYP3A4, CYP3A5, CYP2C9) by RT-PCR in 10 different brain tumor samples. Because P450 may be downregulated by interleukin-1 (IL-1) and IL-6, the receptors for IL-1 and IL-6 were analyzed. None of the brain tumors was positive for CYP3A4 whereas CYP3A5 was detected in 3 out of 10 tumors (two meningeomas, one medulloblastoma grade IV). All five gliomas, an ependymoma, and a lymphoma-metastase gave no signal. CYP2C9 mRNA was present in every sample studied. All samples were positive for IL-1 and IL-6 receptors. In summary, we have demonstrated that tumors of the CNS express P450, indicating that activation of prodrugs like oxazaphosphorines may take place intratumorally. However, the most abundantly hepatically expressed CYP3A4 enzyme is absent in the brain tumor samples. The presence of the IL-1 and IL-6 receptors opens the possibility that the wellknown downregulating influence of these cytokines also takes place in brain tumors.     

P‐glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel

Paclitaxel is avidly transported by P‐glycoprotein (P‐gp/MDR1/ABCB1). This results in low oral bioavailability, which can be boosted by coadministration of P‐gp inhibitors. Unlike paclitaxel, docetaxel is extensively metabolized by CYP3A4 and its oral bioavailability can be enhanced in mice and humans by coadministration of the potent CYP3A inhibitor ritonavir. Unexpectedly, ritonavir also enhances the oral bioavailability of paclitaxel in humans. We aimed to resolve the mechanism underlying this enhancement. Using mice lacking Cyp3a and/or P‐gp, we investigated the combined and separate restricting roles of Cyp3a and P‐gp in the oral bioavailability of paclitaxel, and the boosting effect of ritonavir. CYP3A4‐humanized mice were used for translation to the human situation. P‐gp had a dominant effect (11.6‐fold, p < 0.001) over Cyp3a (<1.5‐fold, n.s.) in limiting plasma concentrations of oral paclitaxel. However, in the absence of P‐gp, Cyp3a decreased paclitaxel plasma concentrations twofold (p < 0.001). Coadministered ritonavir inhibited Cyp3a‐mediated metabolism, but not P‐gp‐mediated transport of paclitaxel. Owing to the dominant effect of P‐gp, ritonavir enhanced only paclitaxel plasma concentrations in P‐gp‐deficient mice. Mouse liver microsomes metabolized paclitaxel far less efficiently than human or CYP3A4‐transgenic liver microsomes, revealing much lower efficiency of paclitaxel metabolism by mouse than by human CYP3As. Accordingly, ritonavir could enhance the oral bioavailability of paclitaxel in CYP3A4‐humanized mice, despite the fact that these mice are P‐gp‐proficient. Our results show that CYP3A4 inhibition most likely underlies the boosting effect of ritonavir on oral paclitaxel bioavailability in humans. Furthermore, CYP3A4‐humanized mice allow improved understanding of CYP3A4‐mediated paclitaxel metabolism in humans.     

Expression of high-affinity interleukin 4 receptors on murine sarcoma cells and receptor-mediated cytotoxicity of tumor cells to chimeric protein between interleukin 4 and Pseudomonas exotoxin

The presence of interleukin 4 receptor (IL-4R) on methylcholanthrene (MCA-106, MCA-102, and MC-38)- and viral DNA (G-2TS and 14-2TS)-induced murine sarcoma cells was demonstrated. MCA-106 tumor cells express about 500 to 1348 (median, 800) interleukin 4 (IL-4) binding sites/cell with a dissociation constant (Kd) of 115 +/- 26 pM (mean +/- SD, n = 4). By Northern blot analysis, tumor cells exhibited a single mRNA species of 3.9 kilobases. Other murine sarcoma (MCA-102), colon adenocarcinoma (MC-38), G-2TS, and 14-2TS tumor cells express low numbers of IL-4R. By immunoperoxidase staining, 81 to 92% of the cells from fresh MCA-106 tumors were positive for IL-4 receptors, while only 7 to 10% of tumor-infiltrating cells were Thy 1.2 and less than 1% Mac-1 positive. Using a chimeric protein composed of IL-4 and Pseudomonas exotoxin (IL-4-PE40), we observed that IL-4-PE40 was cytotoxic (determined by inhibition of protein synthesis by [3H]leucine uptake) to MCA-106 tumor cells in a dose-dependent manner. A nonchimeric protein (PE40) that cannot bind to the IL-4R did not inhibit protein synthesis in tumor cells. A chimeric mutant protein (IL4-PE40 asp553) that can bind to IL-4 receptors but does not have the capability to inhibit protein synthesis was not cytotoxic to tumor cells. These studies strongly suggest that IL-4R on murine MCA-106 sarcoma cells is internalized when occupied by IL-4 PE40. Furthermore, a neutralizing antibody (11B11) to IL-4 completely abolished the protein synthesis-inhibitory activity of IL-4-PE40. G-2TS tumor cells which expressed low numbers of IL-4 receptors were not vulnerable to cytotoxicity by IL-4-PE40. Taken together, these data suggest that IL-4 receptor may be a target for IL-4-toxin therapy.     

Cytochrome P450 2a of nasal epithelium: Regulation and role in carcinogen metabolism

In this study, we found that rat nasal coumarin-7–hydroxylase (a) activity was two orders of magnitude higher than rat hepatic COH activity and could be induced by adding coumarin to the rats' drinking water. In western blot analysis, an anti-cytochrome P450 (a) 2a-5 (mouse liver COH) antibody recognized a sharp band in the microsomal fraction of rat nasal epithelium but not of the liver; the band comigrated with Cyp2a-5. The intensity of the band was increased by the coumarin treatment. Similarly, in northern blot analysis, a cDNA probe specific for Cyp2a-5 recognized an mRNA in the nasal epithelium having the same size as mouse liver Cyp2a-5 mRNA; however, no hybridizable mRNA was recognized in liver preparations. Unlike the protein level, the level of the mRNA was not increased by coumarin. When northern blot analyses were performed with two oligoprobes specific for rat lung CYP2A3, an mRNA of similar size to Cyp2a-5 mRNA was recognized. In immunoinhibition analysis, anti-Cyp2a-5 antibody inhibited rat nasal COH activity and aflatoxin B₁ (AFB₁) metabolism completely. It inhibited N-nitrosodiethylamine (a) and 4- (a) -1-(3-pyridyl)-1-butanone (a) metabolism by 80–90%. In contrast, the hepatic metabolism of the four compounds was not affected by the antibody. When coumarin instead of anti-Cyp2a-5 antibody was used, a strong but variable inhibition of the nasal metabolism of AFB₁, NDEA, and NNK was seen. The results suggest that an enzyme or enzymes similar to mouse liver Cyp2a-5, one of which may be CYP2A3, is expressed at high levels in rat nasal epithelium but not in the liver and that its expression is increased by coumarin, an odorant and a substrate of Cyp2a-5. The increase probably occurs by protein stabilization or stimulation of translation. The results also show that the enzyme has a key role in the nasal metabolism of three well-known carcinogens, AFB₁, NDEA, and NNK and may therefore be an important contributing factor in nasal carcinogenesis.©1995 Wiley-Liss, Inc.     

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 (CYP3A4) in increasing the metabolism of AQ4N using a gene-directed enzyme prodrug therapy (GDEPT) strategy. RIF-1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF-1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP-activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation-resistant and chemo-resistant hypoxic tumor cells.     

Tumor-targeted gene transfer in vivo via recombinant Newcastle disease virus modified by a bispecific fusion protein

Previously we have demonstrated that a recombinant Newcastle disease virus (NDV) carrying the transgene EGFP can be retargeted to IL-2 receptor positive tumor cells by a bispecific fusion protein alphaHN-IL-2 in vitro. The purpose of the present study was to investigate the specificity and efficiency of gene delivery to tumor cells in vivo via this modified RNA virus. Prior ex vivo infection of murine lymphoma cells by the modified virus resulted in selective EGFP expression in IL-2R+ target tumor cells in vivo. Direct fluorescence microscopy and immunohistology showed viral replication in target positive tumor tissue resulting in much more EGFP expression than in target negative tumor tissue, 24 h after intratumoral injection of the alphaHN-IL-2 modified NDV. A quantitative real-time RT-PCR for EGFP mRNA. confirmed the selective gene expression in IL-2R+ tumor cells. Biodistribution studies showed that EGFP transgene delivery was reduced by 35-100% in liver, spleen, kidney, lung and thymus by the modified virus, while 98% of the transgene was delivered to IL-2R+ tumors. In conclusion, the modification of NDV by the bispecific protein does not compromise severely the efficiency of gene delivery into IL-2R-positive tumors, but greatly reduces viral gene expression in IL-2R-negative tumors and in normal tissues.     

The effect of an individual's cytochrome CYP3A4 activity on docetaxel clearance.

Docetaxel is a chemotherapeutic agent effective in the treatment of various solid tumors. Patients given a standard dose of docetaxel exhibit wide interpatient variation in clearance (CL) and toxic effects. Docetaxel undergoes metabolism by cytochrome CYP3A4. Thus, interpatient variability in CYP3A4 activity may account in part for differences in toxicity and CL. Twenty-one heavily pretreated patients with metastatic sarcomas received docetaxel (100 mg/m2). Hepatic CYP3A4 activity in each patient was measured by the [14C-N-methyl]erythromycin breath test (ERMBT). Blood samples were taken at selected times over the next 24 h for pharmacokinetic analysis. Phenotypic expression of hepatic CYP3A4 activity measured by the ERMBT varied over 20-fold (administered 14C exhaled in 1 h: mean, 2.53%; range, 0.25-5.35%), which is similar to a normal control population. CL of docetaxel varied nearly 6-fold (mean, 21.0 liters/h/m2; range, 5.4-29.1 liters/h/m2). The ERMBT was the best predictor of CL when compared with serum alanine aminotransferase, albumin, alkaline phosphatase, or serum alpha-1-acidic glycoprotein. The natural log of ERMBT accounted for 67% of the interpatient variation in CL. Multivariate analysis showed that the natural log of ERMBT and albumin together accounted for 72% of the interpatient variation in CL. The greatest toxicity was seen in patients with the lowest ERMBT. Hepatic CYP3A4 activity is the strongest predictor of docetaxel CL and accounts for the majority of interpatient differences in CL. Patients with low CYP3A4 activity are at risk for having decreased CL and may thus experience increased toxicity from docetaxel. Those with high activity may be receiving a suboptimal dose. By measuring CYP3A4 activity, the ERMBT may be clinically useful in tailoring doses of CYP3A4 substrates, such as docetaxel, in certain individuals.     

Ritonavir inhibits intratumoral docetaxel metabolism and enhances docetaxel antitumor activity in an immunocompetent mouse breast cancer model

Docetaxel (Taxotere^®) is currently used intravenously as an anticancer agent and is primarily metabolized by Cytochrome P450 3A (CYP3A). The HIV protease inhibitor ritonavir, a strong CYP3A4 inhibitor, decreased first‐pass metabolism of orally administered docetaxel. Anticancer effects of ritonavir itself have also been described. We here aimed to test whether ritonavir co‐administration could decrease intratumoral metabolism of intravenously administered docetaxel and thus increase the antitumor activity of docetaxel in an orthotopic, immunocompetent mouse model for breast cancer. Spontaneously arising K14cre;Brca1^F/F;p53^F/F mouse mammary tumors were orthotopically implanted in syngeneic mice lacking Cyp3a (Cyp3a^?/?) to limit ritonavir effects on systemic docetaxel clearance. Over 3 weeks, docetaxel (20 mg/kg) was administered intravenously once weekly, with or without ritonavir (12.5 mg/kg) administered orally for 5 days per week. Untreated mice were used as control for tumor growth. Ritonavir treatment alone did not significantly affect the median time of survival (14 vs. 10 days). Median time of survival in docetaxel‐treated mice was 54 days. Ritonavir co‐treatment significantly increased this to 66 days, and substantially reduced relative average tumor size, without altering tumor histology. Concentrations of the major docetaxel metabolite M2 in tumor tissue were reduced by ritonavir co‐administration, whereas tumor RNA expression of Cyp3a was unaltered. In this breast cancer model, we observed no direct antitumor effect of ritonavir alone, but we found enhanced efficacy of docetaxel treatment when combined with ritonavir. Our data, therefore, suggest that decreased docetaxel metabolism inside the tumor as a result of Cyp3a inhibition contributes to increased antitumor activity.     

Delineation of the interactions between the chemotherapeutic agent eribulin mesylate (E7389) and human CYP3A4

PURPOSE: Eribulin mesylate (E7389), a structurally simplified, synthetic analog of the marine natural product halichondrin B, acts by inhibiting microtubule dynamics via mechanisms distinct from those of other tubulin-targeted agents. Eribulin is currently in Phase III clinical trials for the treatment of metastatic breast cancer. Since drug-induced modulation of cytochrome P450 enzymes, particularly CYP3A4, is a frequent cause of drug-drug interactions, we examined the effects of eribulin on the activity and expression of hepatic and recombinant CYP3A4 (rCYP3A4) in vitro. METHODS: Identification of the enzyme(s) responsible for eribulin metabolism was based on compound depletion and metabolite formation in reaction mixtures containing subcellular liver fractions or primary human hepatocytes, plus recombinant Phases I and II metabolic enzymes. The role of the enzyme(s) identified was confirmed using enzyme-selective inhibitors and the correlation with prototypic enzyme activity. The effect of eribulin on enzymatic activity was characterized using both microsomal preparations and recombinant enzymes, while the possible modulation of protein expression was evaluated in primary cultures of human hepatocytes. RESULTS: Eribulin was primarily metabolized by CYP3A4, resulting in the formation of at least four monooxygenated metabolites. In human liver microsomal preparations, eribulin suppressed the activities of CYP3A4-mediated testosterone and midazolam hydroxylation with an apparent K (i) of approximately 20 muM. Eribulin competitively inhibited the testosterone 6beta-hydroxylation, nifedipine dehydration, and R-warfarin 10-hydroxylation activities of rCYP3A4, with an average apparent K (i) of approximately 10 muM. These inhibitions were reversible, with no apparent mechanism-based inactivation. Eribulin did not induce the expression or activities of CYP1A and CYP3A enzymes in human primary hepatocytes, and clinically relevant concentrations of eribulin did not inhibit CYP3A4-mediated metabolism of various therapeutic agents, including carbamazepine, diazepam, paclitaxel, midazolam, tamoxifen, or terfenadine. CONCLUSIONS: Eribulin was predominantly metabolized by CYP3A4. Although eribulin competitively inhibited the testosterone 6beta-hydroxylation, nifedipine dehydration, and R-warfarin 10-hydroxylation activities of rCYP3A4, it did not induce or inhibit hepatic CYP3A4 activity at clinically relevant concentrations. As eribulin does not appear to affect the metabolism of other therapeutic agents by CYP3A4, our data suggest that eribulin would not be expected to inhibit the metabolism of concurrently administered drugs that are metabolized by CYP3A4, suggesting a minimal risk of drug-drug interactions in the clinical setting.     

7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.     

Cytochrome P450 isozymes 3A4 and 2B6 are involved in the in vitro human metabolism of thiotepa to TEPA

PURPOSE: To establish the cytochrome P450 (CYP) isozymes involved in the metabolism of the alkylating agent, thiotepa, to the pharmacologically active metabolite, TEPA.METHODS: In vitro chemical inhibition studies were conducted by incubating thiotepa and pooled human hepatic microsomes in the presence of known inhibitors to CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Studies were also performed with cloned, expressed CYP3A4, CYP2A6, CYP2E1 and CYP2B6 microsomes, and anti-CYP2B6 monoclonal antibody.RESULTS: Known CYP3A4 inhibitors reduced TEPA production. Inhibition with CYP2E1 inhibitors was inconsistent. All other inhibitors produced little or no change in TEPA formation. Cloned, expressed CYP2B6 and CYP3A4 microsomes catalyzed TEPA formation, whereas CYP2A6 and CYP2E1 did not. Incubation of thiotepa with anti-CYP2B6 antibody and cloned, expressed CYP2B6 microsomes resulted in reductions in the formation of TEPA, but no change in TEPA formation occurred in human liver microsomes.CONCLUSIONS: Thiotepa is metabolized in human liver microsomes by CYP3A4 (major) and CYP2B6 (minor). There is a potential for CYP-mediated drug interactions with thiotepa. Pharmacokinetic variability of thiotepa may be related to expression of hepatic CYP isozymes.     

Role of Cyp1A1 in modulation of antitumor properties of the novel agent 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) in human breast cancer cells

2-(4-Amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) is a candidate antitumor agent with potent and selective activity against human-derived tumor cell lines in vitro and in vivo. Only sensitive cell lines (e.g., MCF-7) were able to accumulate and metabolize DF 203, forming the main inactive metabolite, 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole (6-OH 203). Selective metabolism may therefore underlie its antitumor profile. DF 203 6-hydroxylase activity by MCF-7 cells was not constitutive but induced only after pretreatment of cells with DF 203, 3-methylcholanthrene, or beta-naphthoflavone. 6-Hydroxylation was strongly inhibited by either goat antirat cytochrome P450 1A1 (CYP1A1) serum or alpha-naphthoflavone. Both alpha-naphthoflavone and 6-OH 203 abrogated DF 203-induced growth inhibition. Microsomes from genetically engineered human B-lymphoblastoid cells expressing CYP1A1, CYP1B1, or CYP2D6 metabolized DF 203 to 6-OH 203. Immunoblot analysis detected significantly enhanced CYP1A1 protein in a panel of sensitive breast cancer cell lines after exposure to DF 203. Neither constitutive expression nor induction of CYP1A1 protein was detected in nonresponsive breast (HBL 100, MDA-MB-435, and MCF-7/ADR) and prostate (PC 3 and DU 145) cancer cell lines. The expression of CYP1B1 was also modulated by DF 203 in the same sensitive cell lines. However, of the two isoforms, only CYP1A1 activity was irreversibly inhibited by DF 203 and significantly inhibited by 6-OH 203. In sensitive cell lines only, [14C]DF 203-derived radioactivity bound covalently to a Mr 50,000, protein which was immunoprecipitated by CYP1A1 antiserum. The covalent binding of [14C]DF 203 to recombinant CYP1A1 enzyme was NADPH-dependent and reduced by 6-OH 203 and glutathione. CYP1A1 appears essential for the metabolism of DF 203 and may have a pivotal, yet undefined, role in its antitumor activity.     

WHV＼myc转基因小鼠肝癌发生过程中c—myc基因表达的双相性

目的：WHV／myc转基因小鼠其肝脏肿瘤的自然发生率很高，本研究旨在细胞水平探测WHV／myc转基因小鼠肝癌发生过程中。c-myc转染基因的表达方式和肝细胞的增殖活性。方法：用核酸分子原位杂交的方法，检测了WHV／myc转基因小鼠肝肿瘤形成的不同阶段肝组织中c-myc基因的表达，同时检测了细胞增殖标志-组蛋白H3-2mRNA的表达。结果：10天龄小鼠肝脏中c-myc转染基因呈中等程度的表达，伴随30％的肝细胞增殖。随后，该基因表达水平迅速降低，在2月龄、4月龄和9月龄小鼠的肝脏中均不能够检测出。此期间肝细胞的增殖活性亦处于低水平。c-myc转染基因的表达重新出现于肿瘤形成期，肝腺瘤和肝癌的表达方式和强度相仿。肝细胞的增殖活性在此期约为14％左右。结论：c-myc转染基因在小鼠出生后早期和肿瘤形成期的异常表达对肝肿瘤的发生和瘤细胞转化表型的维持可能具有重要意义。     

Cell-density-dependent Expression of Cyp1a2 Gene in Monolayer-cultured Adult Mouse Hepatocytes

Expression of Cyp 1a1 and Cyp 1a2 genes was investigated in adult C57BL/6NCrj mouse hepatocytes for up to 5 days after transfer to monolayer culture. CYP1A1 mRNA was substantially induced by treatment with 3-methylcholanthrene during the observation period, independently of the seeded cell density. However, expression of CYP1A2 mRNA was dependent on cell density and was higher in cells cultivated at lower density. With increasing culture period the expression was decreased, so that only negligible levels were evident by day 5, and reduced expression of constitutive and induced CYP1A2 mRNA became apparent earlier in more densely seeded cells. This was not related to differences in numbers of inducer molecules per cell. While mouse hepatocytes incorporated tritium-labeled thymidine under the given culture conditions, induction of expression of the two Cyp 1a genes did not show any direct relationship with DNA synthesizing activity. These observations suggest some role for Cyp 1a2 during changes in physiological state.     

Differences in the hepatic P450-dependent metabolism of estrogen and tamoxifen in response to treatment of rats with 3,3'-diindolylmethane and its parent compound indole-3-carbinol

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo product 3,3'-diindolylmethane (DIM), have been reported to suppress estrogen-responsive cancers. This effect may be mediated through the modification of cytochrome P450 (CYP) complement and activities leading to estrogen detoxification. We examined the effects of a 4-day treatment of female Sprague-Dawley rats with DIM at 8.4 and 42 mg/kg body weight (bwt), on the hepatic CYP protein level, CYP1A1, 1A2, 2B1/2 and 3A1/2 probe activities and CYP-dependent metabolism of 17beta-estradiol (E2) and estrone (E1). At 42 mg/kg bwt, DIM effected a small increase (2.8-fold) in CYP1A1 activity, and at both dose levels it reduced CYP3A1/2 activity by approximately 40%. At the higher dose level, DIM decreased the rates of oxidation of E2 to 4-OH-E2, 4-OH-E1, 6alpha-OH-E2 and 6(alpha+beta)-OH-E1 by 39, 44, 71 and 60%, respectively, and E1 to 6(alpha+beta)-OH-E1 by 39%. These effects were considerably different from those of I3C reported by us previously. We also examined the effects of DIM and I3C on the hepatic microsomal metabolism of tamoxifen (TAM). Whereas metabolism of TAM was unaffected by DIM, formation of N-desmethyl-TAM (and its presumed derivative) was increased approximately 3-fold by I3C at 250 mg/kg bwt. Since N-desmethyl-TAM is transformed to a genotoxic metabolite, dietary exposure to I3C may enhance hepatic carcinogenicity of TAM in the rat. The differences between I3C and DIM in CYP-mediated activities and metabolism indicate that DIM is not a proximate intermediate in the mechanism of action of I3C.     

Differences in regulation of gene expression between Cyp1a-1 and Cyp1a-2 in adult mouse hepatocytes in primary culture.

The regulation of expression of Cyp1a-1 and Cyp1a-2 genes was investigated in adult C57BL/6NCrj mouse hepatocytes for up to 5 days after transferring to either monolayer or spheroid (multi-cellular aggregate) primary culture. The expression of 3-methylcholanthrene (MCA)-induced CYP1A1 mRNA remained high during the observation period under both monolayer and spheroid culture conditions. In contrast, while levels of CYP1A2 mRNA in spheroid culture were also appreciable throughout, they rapidly decreased in monolayer culture to become negligible. An increase in intracellular cyclic nucleotide content induced CYP1A1 mRNA in the later culture period in either spheroid or monolayer cultures. A significant elevation of both basal and MCA-induced 7-ethoxycoumarin-O-deethylase, and MCA-induced aryl hydrocarbon hydroxylase and 7-methoxyresorufin-O-demethylase activities was observed in the presence of intracellular cyclic nucleotide content-increasing agents, although the extent of enhancement far exceeded that expected from the scarce changes in MCA-induced CYP1A1 mRNA levels. Basal and MCA-induced CYP1A2 mRNA expression were not changed by altering intracellular cyclic nucleotide content. The level of CYP1A1 mRNA after MCA treatment was elevated in the presence of cycloheximide. Furthermore, with increasing culture time, addition of this agent caused expression of Cyp1a-1 gene in MCA-untreated cells. In contrast, the presence of cycloheximide did not increase constitutive or MCA-induced CYP1A2 mRNA. These observations indicate that expression of Cyp1a-1 and Cyp1a-2 genes may be regulated by different mechanisms.