Search

Aldehyde dehydrogenase (ALDH) is well known for its involvement in the resistance of tumor cells to cyclophosphamide (CPA) and its activated derivatives, such as 4-hydroperoxy-CPA (4HC). The role of other drug-metabolizing enzymes such as glutathione S-transferase (GST) in CPA resistance is, however, less certain. In the present study of a human breast cancer cell line (MCF-7) exhibiting about 6-fold resistance to 4HC (MCF/HC), cellular levels of glutathione (GSH) were increased 1.4-fold, while cytosolic GST and ALDH activities were increased 2.7- and 7.2-fold, respectively, relative to the MCF-7 parental line. No significant changes in glutathione peroxidase and NADPH cytochrome P450 reductase activity, and no increase in microsomal GST and GST pi mRNAs were found in the resistant cells. Treatment with the ALDH substrate octanal sensitized the cells to the cytotoxic effects of 4HC to a modest extent in both MCF-7 and MCF/HC cells [dose modification factor (DMF) of 1.4 and 1.6, respectively]. Depletion of GSH by treatment with the GSH synthesis inhibitor buthionine sulfoximine (BSO) enhanced the cytotoxic effect of 4HC to a similar extent in both cell lines. By contrast, ethacrynic acid, which inhibited GST activity by > 85% in MCF-7 and MCF/HC cell extracts without depletion of GSH, sensitized the resistant but not the parental cells to 4HC cytotoxicity, indicating the importance of GST as a determinant of 4HC resistance in these cells. This conclusion is supported by the observation that in MCF/HC cells, ethacrynic acid in combination with BSO increased the DMF 3-fold higher than did BSO or EA alone, while in the parental MCF-7 cells ethacrynic acid with BSO had no significant chemosensitization effect over BSO alone. These studies establish that in addition to ALDH, GST overexpression can contribute to acquired resistance of tumor cells to 4HC and, furthermore, suggest that modulators that target the GSH/GST system could be useful in overcoming CPA resistance in the clinic.

Metronomic chemotherapy, in which cytotoxic drugs are delivered at low doses at regular, intermittent intervals, has shown promising clinical results in several cancers and offers the benefit of reduced patient toxicity compared to conventional maximum tolerated dose chemotherapy. We previously showed that cyclophosphamide (CPA) treatment on a 6-day repeating metronomic schedule regresses implanted gliomas by an immune-mediated mechanism. Here, we examine whether this immune-stimulatory metronomic chemotherapy can be enhanced by combination with TLR9-stimulating CpG-1826 immunotherapy in GL261 gliomas implanted in immunocompetent BL6 mice. CpG-1826 treatment activated an anti-tumor immune response in ~50% of mice, associated with a strong increase in tumor-associated macrophages. CpG-1826 was ineffective against GL261 tumors implanted in T cell- and B cell-deficient scid mice, suggesting that adaptive immunity is essential. Combination of CpG-1826 with metronomic CPA sensitized GL261 gliomas in both CpG-1826-responsive and CpG-1826-non-responsive mice, increasing infiltration of tumor-associated macrophages, dendritic cells, and T cells and increasing anti-tumor activity. Further, the combination treatment elicited long-term regression and immune memory in a subset of glioma-bearing mice. To identify potential mechanisms for this enhanced response to the combination therapy, we assayed a number of immune-related genes in the treated tumors, including feedback inhibitory immunosuppressive T cell factors that are induced by metronomic CPA treatment. Combination of CpG-1826 with metronomic CPA failed to induce several of these factors, suggesting that TLR9-based immunotherapy can enhance the anti-tumor activity of metronomic CPA by interdicting feedback immune inhibition mechanisms. Grant support: CA49248 (to DJW). Citation Format: Marie Jordan, David J. Waxman. Low dose, metronomic cyclophosphamide therapy sensitizes tumors to CpG-1826 immunotherapy in a preclinical glioma model. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B121.

The growth hormone-regulated transcription factors STAT5 and BCL6 coordinately regulate sex differences in mouse liver, primarily through effects in male liver, where male-biased genes are upregulated and many female-biased genes are actively repressed. Here we investigated whether CUX2, a highly female-specific liver transcription factor, contributes to an analogous regulatory network in female liver. Adenoviral overexpression of CUX2 in male liver induced 36% of female-biased genes and repressed 35% of male-biased genes. In female liver, CUX2 small interfering RNA (siRNA) preferentially induced genes repressed by adenovirus expressing CUX2 (adeno-CUX2) in male liver, and it preferentially repressed genes induced by adeno-CUX2 in male liver. CUX2 binding in female liver chromatin was enriched at sites of male-biased DNase hypersensitivity and at genomic regions showing male-enriched STAT5 binding. CUX2 binding was also enriched near genes repressed by adeno-CUX2 in male liver or induced by CUX2 siRNA in female liver but not at genes induced by adeno-CUX2, indicating that CUX2 binding is preferentially associated with gene repression. Nevertheless, direct CUX2 binding was seen at several highly female-specific genes that were positively regulated by CUX2, including A1bg, Cyp2b9, Cyp3a44, Tox, and Trim24. CUX2 expression and chromatin binding were high in immature male liver, where repression of adult male-biased genes and expression of adult female-biased genes are common, suggesting that the downregulation of CUX2 in male liver at puberty contributes to the developmental changes establishing adult patterns of sex-specific gene expression.

Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient’s tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity.

Sex differences in drug metabolism are recognized as a major determinant of sex differences in pharmacokinetics and an important contributor to interindividual differences in drug metabolism and, in some cases, drug action. Sex differences in human drug metabolism have also been observed with a variety of drugs. The biochemical basis of sex differences in hepatic drug metabolism was largely unknown until the 1980s when it was discovered that the expression of many drug-metabolizing enzymes, including several members of the cytochrome P450 (CYP) superfamily, is sex dependent in rat and mouse liver. Well-studied examples include rat enzymes CYP2C11, which is male specific in its expression, and CYP2C12 (female specific) and CYP2A1 (female predominant). The major gonadal hormones, testosterone and estradiol, regulate the sex-dependent expression of rat hepatic CYP enzymes indirectly, through their effects on the hypothalamus and its regulation of the sexually dimorphic, ultradian rhythm of pituitary growth hormone (GH) secretion, which ultimately dictates the sex-dependent patterns of expression of these CYPs and certain other drug-metabolizing enzymes. In male rats, the intermittent, pulsatile pattern of GH secretion stimulates the expression of male-specific liver enzymes, whereas the more continuous pattern of GH release in females suppresses the expression male-specific enzymes while inducing female-specific enzymes. The current view is that the transcription factors STAT5b and HNF4α coordinately regulate the action of GH and its resultant effect on the sex-dependent expression of hepatic CYP genes and thus provide a molecular basis for sex differences in drug metabolism. Keywords: sex differences; hormonal regulation; cytochrome P450; growth hormone; gonadal hormones

The role of steroids related to the adrenal androgen dehydroepiandrosterone (5-androstene-3 beta-ol-17-one; DHEA) in regulating the expression of peroxisomal and cytochrome P-450 4A (CYP4A) enzymes active in fatty acid metabolism was assessed using a primary rat hepatocyte culture system. Exposure of hepatocytes to the peroxisome proliferator, clofibric acid (10-250 microM), for 48-96 h led to substantial increases in CYP4A protein, CYP4A1, CYP4A2 and CYP4A3 mRNAs, and the mRNAs encoding both forms of peroxisomal acyl-CoA oxidase (ACOX-I and ACOX-II), as judged by Northern-blot analysis using gene-specific oligonucleotide probes. Although DHEA treatment in vivo is effective in inducing these mRNAs in rat liver, it had no effect in the cultured hepatocytes. In contrast, treatment of the cells with DHEA 3 beta-sulphate (DHEA-S; 10-250 microM) stimulated major increases in CYP4A and ACOX mRNA levels. Examination of several analogues indicated a preference for 3 beta-sulphate over 17 beta-sulphated steroids and the inactivity of a 3 alpha-hydroxy-17 beta-sulphate derivative (DHEA-S > 5-androstene-3 beta,17 beta-diol 3-sulphate approximately 5 alpha-androstene-3 beta-ol-17-one 3-sulphate > 5-androstene-3 beta, 17 beta,17 beta-diol 17-sulphate approximately 5 beta-androstane-3 alpha-ol-17-one 3-sulphate >> 5 alpha-androstane-3 alpha, 17 beta-diol 17-sulphate). Induction of CYP4A mRNAs by either DHEA-S or clofibric acid was partially blocked by structurally diverse Ca(2+)-channel antagonists (nicardipine, nifedipine and diltiazem; 50 microM), suggesting that both the steroidal and fibrate classes of CYP4A inducers stimulate peroxisomal-proliferative responses via a Ca(2+)-dependent pathway. Retinoic acid alone slightly induced CYP4A mRNAs but did not enhance the induction by clofibrate or DHEA-S. As DHEA-S corresponds to a physiologically important major circulating androgen, these findings suggest that it may serve as an endogenous regulator of hepatic peroxisome enzyme levels. They further suggest that Ca(2+)-channel blockers may be useful pharmacological tools for the further study of the underlying cellular mechanism whereby endogenous steroids and fibrate drugs induce peroxisome proliferation, and the relationship of these events to activation of the peroxisome proliferator-activated receptor.

The cytochrome P450 2B subfamily in the rat contains an estimated eight to eleven members at the genomic level. Synthesis in the liver of the prototypic forms P450 2B1 and P450 2B2 is dramatically induced by phenobarbital. The 1.9-kb mRNA for P450 2B3, a third member of the P450 2B subfamily, is constitutively present in rat liver but is not inducible by phenobarbital. We have now cloned and sequenced exonic sequences corresponding to the entire 2B3 mRNA and determined their exon-intron structure, which is identical to that of CYP2B1/CYP2B2 and other CYP2B genes. A putative CYP2B3 transcription start site was identified and CYP2B3 5'- and 3'-flanking sequences were compared to those of CYP2B1 and CYP2B2. CYP2B3, like CYP2B1 and CYP2B2, has a modified TATA box preceding the transcription start site and lacks the canonical polyadenylation signal preceding the poly(A) site. A 2B3 expression vector, pMT2-2B3, directed the synthesis in COS-1 cells of an approximately 50-kD protein detectable on Western blots with a polyclonal antibody and with one of four monoclonal antibodies raised against 2B1 but not with a polyclonal antibody raised against P450 PB6. The 2B3 protein migrated with a slightly higher electrophoretic mobility than 2B1 and comigrated with a protein detected by anti-2B1 antibodies in liver microsomes from untreated rats. The results indicate that a 2B3-like protein is present in rat liver and that it is distinct from P450 PB6 and other known constitutive rat hepatic P450s.

Most malignant tumors of the central nervous system do not respond well to chemotherapy. The anticancer drug cyclophosphamide (CPA) is largely ineffective against these neoplasms as its conversion to DNA-alkylating, cytotoxic metabolites is restricted primarily to the liver and these metabolites do not readily cross the blood-brain barrier. Here, we show that brain tumor cells can be sensitized to the cytotoxic effects of CPA, both in culture and in vivo, by introduction of the hepatic enzyme responsible for the activation of CPA, cytochrome P450 2B1. Stable transfection of rat C6 glioma cells with the P450 2B1 gene rendered the cultured tumor cells sensitive to CPA. Further, C6 cells bearing this gene were more sensitive than parental cells to the cytotoxic action of CPA when grown subcutaneously in the flanks of athymic mice. Murine fibroblasts producing a retrovirus vector encoding P450 2B1 and expressing this enzyme were then prepared and grafted into the brains of athymic mice seeded with rat C6 gliomas. Intrathecal administration of CPA prevented the development of meningeal neoplasia and led to partial regression of the parenchymal tumor mass. By contrast, C6 glioma-bearing mice receiving fibroblasts expressing the Escherichia coli lacZ gene and CPA exhibited extensive meningeal tumors and parenchymal solid brain tumors. The in situ activation of CPA by cytochrome P450 2B1 provides a novel approach not only for brain tumor gene therapy, but also for negative, drug-conditional selection of other defined cell populations.

Sexually dimorphic regulation of phenobarbital-induced cytochromes P450 2B1 and 2B2 (collectively referred to as P450 2B) as well as P450 2B-dependent monooxygenase activities was studied in multi-hormone-depleted hypophysectomized rats and in growth hormone (GH)-deficient monosodium glutamate (MSG)-treated rats. Our results indicate that endogenous GH suppresses phenobarbital induction of P450 2B and that the feminine pattern of continuous GH secretion is more suppressive than the masculine profile of episodic secretion. Moreover, we have found that it is the height of the GH pulse, and not necessarily its frequency nor the interpulse trough periods, that signals the suppressive effects of GH on P450 2B expression in male rats. Last, irrespective of the presence or absence of circulating GH, the magnitude of phenobarbital induction of P450 2B and associated monooxygenases was consistently lower in female rats, suggesting the presence of some degree of sex-dependent, but GH-independent regulation.

We evaluated the expression of interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta mRNAs in the intragastric-feeding rat model of alcoholic liver disease. Rats were fed different diets for periods of 2 or 4 wk. Animals fed saturated fat and ethanol and the corn oil-dextrose control group had no liver injury, whereas animals fed corn oil and ethanol showed pathologic changes. RNA was extracted from the livers at the time of killing, reverse-transcribed and amplified; polymerase chain reaction products were subjected to electrophoresis on agarose gel. Interleukin-1 alpha mRNA was present in all groups at 2 and 4 wk; interleukin-1 beta and transforming growth factor-beta mRNAs were present in all groups at 4 wk. Tumor necrosis factor-alpha mRNA was absent in all groups at 2 wk but was present in the corn oil-ethanol group only at 4 wk. Because pathological liver injury was evident in the corn oil-ethanol group by 4 wk, the presence of tumor necrosis factor-alpha mRNA at this time suggests a pathogenetic role for tumor necrosis factor-alpha in alcohol-induced liver injury.

Hepatic cytochrome P450 cholesterol 7 alpha-hydroxylase, CYP7, is regulated in vivo at the protein and the mRNA level in response to multiple physiological factors, including liver cholesterol synthesis, bile acid feedback inhibition, and diurnal rhythm. In the present study we investigated whether the liver transcription factor DBP (albumin promoter D-site binding protein), which undergoes a striking diurnal rhythm in rat liver (DBP levels during evening/morning approximately 100:1), contributes to the diurnal regulation of CYP7 gene expression. DNase I footprinting analysis using bacterially expressed DBP and a cloned 5'-flanking DNA segment of the rat CYP7 gene revealed five distinct DBP-binding sites, designated A-E, distributed between nucleotides (nts) -41 and -295 relative to the CYP7 transcription start site. CYP7-directed gene transcription in HepG2 cells transfected with a 5'-CYP7 promoter-chloramphenicol acetyl-transferase reporter was activated up to 12-fold upon cotransfection of a DBP expression vector, whereas an HNF-1 alpha expression vector did not stimulate CYP7 gene activity. 5'-Deletion analyses and site-specific mutagenesis revealed that this stimulating effect of DBP can in part be ascribed to its functional interaction with DBP binding sites B (nts -115/-125), C (nts -172/-195), and D (nts -214/-230). C/EBP beta (LAP), another liver-enriched basic-leucine zipper transcription factor, bound to these same sites but effected a more modest increase in CYP7-directed gene transcription (up to 3-4-fold) when expressed in HepG2 cells. Competition for CYP7 promoter-binding sites between C/EBP, which undergoes < or = 2-fold diurnal change in rat liver, and the diurnally regulated DBP is proposed to determine the relative rates of basal versus diurnally regulated CYP7 gene transcription and thus may be a primary mechanism for setting the 3-6-fold amplitude that characterizes the circadian rhythm of liver CYP7 expression. Moreover, since DBP is first expressed in rat liver 3-4 weeks after birth, these findings may account for both the enhanced expression and the onset of the diurnal pattern of CYP7 enzyme levels at this stage of development.

Sex-dependent pituitary growth hormone (GH) secretory patterns determine the sex-biased expression of >1,000 genes in mouse and rat liver, affecting lipid and drug metabolism, inflammation, and disease. A fundamental biological question is how robust differential expression can be achieved for hundreds of sex-biased genes simply based on the GH input signal pattern: pulsatile GH stimulation in males versus near-continuous GH exposure in females. STAT5 is an essential transcriptional mediator of the sex-dependent effects of GH in the liver, but the mechanisms that underlie its sex-dependent actions are obscure. Here we elucidate the dynamic, sex-dependent binding of STAT5 and the GH/STAT5-regulated repressor BCL6 to mouse liver chromatin genome wide, revealing a counteractive interplay between these two regulators of sex differences in liver gene expression. Our findings establish a close correlation between sex-dependent STAT5 binding and sex-biased target gene expression. Moreover, sex-dependent STAT5 binding correlated positively with sex-biased DNase hypersensitivity and H3-K4me1 and H3-K4me3 (activating) marks, correlated negatively with sex-biased H3-K27me3 (repressive) marks, and was associated with sex-differentially enriched motifs for HNF6/CDP factors. Importantly, BCL6 binding was preferentially associated with repression of female-biased STAT5 targets in male liver. Furthermore, BCL6 and STAT5 common targets but not BCL6 unique targets showed strong enrichment for lipid and drug metabolism. These findings provide a comprehensive, genome-wide view of the mechanisms whereby these two GH-regulated transcription factors establish and maintain sex differences affecting liver physiology and disease. The approaches used here to characterize sex-dependent STAT5 and BCL6 binding can be applied to other condition-specific regulatory factors and binding sites and their interplay with cooperative chromatin binding factors.

Elastic scattering spectroscopy (ESS), in the form of wavelength-dependent backscattering measurements, can be used to monitor apoptosis in cell cultures. Early changes in backscattering upon apoptosis induction are characterized by an overall decrease in spectral slope and begin as early as 10 to 15 min post-treatment, progressing over the next 6 to 8 h. The timescale of early scattering changes is consistent with reports of the onset of apoptotic volume decrease (AVD). Modeling cellular scattering with a fixed distribution of sizes and a decreasing index ratio, as well as an increased contribution of the whole cell to cellular scattering, resulting from increased cytoplasmic density, is also consistent with observed spectral changes. Changes in ESS signal from cells undergoing osmotically-induced volume decrease in the absence of apoptosis were similar, but smaller in magnitude, to those of apoptotic cells. Further, blockage of Cl(-) channels, which blocks AVD and delays apoptosis, blocked the early scattering changes, indicating that the early scattering changes during apoptosis result, at least partially, from AVD. Work continues to identify the additional sources of early spectral scattering changes that result from apoptosis induction.

ChIP-Seq is widely used to characterize genome-wide binding patterns of transcription factors and other chromatin-associated proteins. Although comparison of ChIP-Seq data sets is critical for understanding cell type-dependent and cell state-specific binding, and thus the study of cell-specific gene regulation, few quantitative approaches have been developed. Here, we present a simple and effective method, MAnorm, for quantitative comparison of ChIP-Seq data sets describing transcription factor binding sites and epigenetic modifications. The quantitative binding differences inferred by MAnorm showed strong correlation with both the changes in expression of target genes and the binding of cell type-specific regulators.

Apoptosis, a form of programmed cell death with unique morphological and biochemical features, is dysregulated in cancer and is activated by many cancer chemotherapeutic drugs. Noninvasive assays for apoptosis in cell cultures can aid in screening of new anticancer agents. We have previously demonstrated that elastic scattering spectroscopy can monitor apoptosis in cell cultures. In this report we present data on monitoring the detailed time-course of scattering changes in a Chinese hamster ovary (CHO) and PC-3 prostate cancer cells treated with staurosporine to induce apoptosis. Changes in the backscattering spectrum are detectable within 10 min, and continue to progress up to 48 h after staurosporine treatment, with the magnitude and kinetics of scattering changes dependent on inducer concentration. Similar responses were observed in CHO cells treated with several other apoptosis-inducing protocols. Early and late scattering changes were observed under conditions shown to induce apoptosis via caspase activity assay and were absent under conditions where apoptosis was not induced. Finally, blocking caspase activity and downstream apoptotic morphology changes prevented late scattering changes. These observations demonstrate that early and late changes in wavelength-dependent backscattering correlate with the presence of apoptosis in cell cultures and that the late changes are specific to apoptosis.

Sex-differences in human liver gene expression were characterized on a genome-wide scale using a large liver sample collection, allowing for detection of small expression differences with high statistical power. 1,249 sex-biased genes were identified, 70% showing higher expression in females. Chromosomal bias was apparent, with female-biased genes enriched on chrX and male-biased genes enriched on chrY and chr19, where 11 male-biased zinc-finger KRAB-repressor domain genes are distributed in six clusters. Top biological functions and diseases significantly enriched in sex-biased genes include transcription, chromatin organization and modification, sexual reproduction, lipid metabolism and cardiovascular disease. Notably, sex-biased genes are enriched at loci associated with polygenic dyslipidemia and coronary artery disease in genome-wide association studies. Moreover, of the 8 sex-biased genes at these loci, 4 have been directly linked to monogenic disorders of lipid metabolism and show an expression profile in females (elevated expression of ABCA1, APOA5 and LDLR; reduced expression of LIPC) that is consistent with the lower female risk of coronary artery disease. Female-biased expression was also observed for CYP7A1, which is activated by drugs used to treat hypercholesterolemia. Several sex-biased drug-metabolizing enzyme genes were identified, including members of the CYP, UGT, GPX and ALDH families. Half of 879 mouse orthologs, including many genes of lipid metabolism and homeostasis, show growth hormone-regulated sex-biased expression in mouse liver, suggesting growth hormone might play a similar regulatory role in human liver. Finally, the evolutionary rate of protein coding regions for human-mouse orthologs, revealed by dN/dS ratio, is significantly higher for genes showing the same sex-bias in both species than for non-sex-biased genes. These findings establish that human hepatic sex differences are widespread and affect diverse cell metabolic processes, and may help explain sex differences in lipid profiles associated with sex differential risk of coronary artery disease.

R.F. receives research funds from the Agence National de la Recherche, the Institut National du Cancer, and the Association for International Cancer Research. Y.L.B. receives basic and clinical research funds from INSERM, the Université Pierre et Marie Curie-Paris 6 (UPMC), the French Research Agency (ANR), the French National Ministry of Health (PHRC), and the Biomedecine Agency. M.Sk., M.Sz., and D.J.W. receive research funding from the United States National Institutes of Health. A.S. receives research funds from the New Zealand government., Hochberg, Z., Feil, R., Constancia, M., Fraga, M., Junien, C., Carel, J.-C., Boileau, P., Le Bouc, Y., Deal, C.L., Lillycrop, K., Scharfmann, R., Sheppard, A., Skinner, M., Szyf, M., Waterland, R.A., Waxman, D.J., Whitelaw, E., Ong, K., Albertsson-Wikland, K.

The alkylating agent BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] can be inactivated through denitrosation reactions catalyzed by both cytosolic and microsomal enzymes. While previous studies have identified a class mu glutathione S-transferase [rat transferase 4-4 (Yb2)] as a major catalyst of the cytosolic denitrosation reaction, the enzymatic catalysts of BCNU denitrosation in microsomal membranes have not been identified. In the present study, both NADPH and glutathione (GSH) were found to support BCNU denitrosation catalyzed by isolated rat liver microsomes. Treatment of rats with the microsomal enzyme inducers phenobarbital and dexamethasone increased NADPH-dependent liver microsomal BCNU denitrosation up to fivefold without major effect on the GSH-dependent denitrosation activity. Although the NADPH-dependent activity was fully inhibited by antibody to NADPH-P450 reductase, purified NADPH-P450 reductase catalyzed BCNU denitrosation at rates that could only account for approximately 2-3% of the microsomal activity. Other experiments, including selective inhibition of NADPH-dependent microsomal BCNU denitrosation by chemical and antibody inhibitors of cytochrome P450, competitive inhibition of P450-catalyzed cyclophosphamide and ifosfamide activation by BCNU, and reconstitution of the denitrosation reaction by purified P450 enzyme 2B1 (major phenobarbital-inducible P450 form), established an important role for cytochrome P450 in BCNU denitrosation. By contrast, GSH-dependent microsomal BCNU denitrosation was unaffected by cytochrome P450 inhibitors, but was inhibited, with varying degrees of selectivity, by the microsomal glutathione S-transferase inhibitors ethacrynic acid, bromosulfophthalein, and indomethacin. These studies establish that BCNU inactivation can be catalyzed by two independent microsomal enzyme systems and suggest that therapeutically useful improvements in BCNU antitumor activity might be achieved through differential inhibition of these enzyme systems in tumor as compared to extratumoral sites.

The sex-dependent expression and growth hormone (GH) regulation of rat liver glutathione S-transferase (GST) was examined using oligonucleotide probes that distinguish between closely related class Alpha (Ya1, Ya2, Yc) and class Mu (Yb1, Yb2, Yb3) GST mRNAs [Waxman, Sundseth, Srivastava and Lapenson (1992) Cancer Res. 52, 5797-5802]. Northern-blot analysis revealed that the steady-state levels of GST Ya1, Yb1 and Yb2 mRNAs are 2.5-3-fold higher in male as compared with female rat liver. In contrast, GST Yc and Ya2 mRNAs were expressed at a 2-3-fold higher level in female rat liver. Microsomal GST mRNA did not exhibit significant sex-dependent differences in rat liver. Treatment of male rats with GH by continuous infusion suppressed expression of the male-dominant GST Ya1, Yb1 and Yb2 mRNAs to levels at or below those found in female rat liver. This suppressive effect of GH was liver-specific, insofar as GH treatment did not alter kidney GST Ya1 mRNA levels. Hypophysectomy increased expression of the male-dominant GSTs, particularly in female rats (e.g. 8-fold elevation of GST Ya1 mRNA). GST Yc mRNA was increased approx. 2-fold in hypophysectomized males, indicating that this mRNA is subject to negative regulation by one or more pituitary-dependent factors. Continuous GH treatment of the hypophysectomized rats suppressed the expression of mRNA of GSTs Ya1, Yb1 and Yb2 when given as a continuous infusion, but not when given by an intermittent (twice daily) GH-injection schedule. Combination of continuous exposure to GH with thyroxine treatment resulted in a more complete suppression of GSTs Ya1, Yb1 and Yb2. In contrast, thyroxine increased the expression of GST Yc in hypophysectomized rats. These studies establish that several Alpha and Mu class GSTs are expressed in a sex-dependent fashion in adult rat liver, where they are regulated by multiple pituitary-dependent hormones through pretranslational mechanisms.

We evaluated the role of changes in microsomal phospholipases (A and C) and arachidonic acid in the intragastric rat feeding model. The experimental animals (male Wistar rats), divided into 4-5 rats/group, were fed the following diets: corn oil and ethanol and corn oil plus dextrose. One set of groups was killed after 2 weeks of feeding, and the second set was killed after 1 month. For each animal, microsomal analysis of cytochrome P-450 2E1 (CYP 2E1) and fatty acids was done. Fourteen animals had analyses of phospholipase C (PLC) and phospholipase A (PLA), and 10 animals had measurements of conjugated dienes. A significant correlation was obtained between the level of CYP 2E1 and the decrease in arachidonic acid (AA) from baseline levels (r = 0.69, p < 0.01). The decrease in AA also correlated with the increase in conjugated dienes (r = 0.70, p < 0.05). PLA and PLC activities were both significantly increased in the corn oil and ethanol groups. The activity of PLC correlated with the decline in AA (r = 0.69, p < 0.01). The correlations noted between the decrease in microsomal AA and CYP 2E1 induction and conjugated diene formation suggest that these processes may be interlinked especially in regard to generation of lipid peroxides that may play a role in alcoholic liver injury.

CYP 3A10 is a hamster liver cytochrome P-450 (P450) that encodes lithocholic acid 6 beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithocholate. Western-blot analysis revealed that the expression of CYP 3A10 protein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not been established whether the specificities of bile acid hydroxylase P450s, such as CYP 3A10, are restricted to their anionic bile acid substrates, we investigated the role of CYP 3A10 in the metabolism of a series of neutral steroid hormones using cDNA directed-expression in COS cells. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6 beta-hydroxylation corresponding to a major activity in all three instances. CYP 3A10-dependent steroid hydroxylation was increased substantially when the microsomes were prepared from COS cells co-transfected with NADPH:P450 reductase cDNA. In this case, the expressed P450 actively catalysed the 6 beta-hydroxylation of testosterone (288 +/- 23 pmol of product formed/min per mg of COS-cell microsomal protein), androstenedione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/min per mg). Other major CYP 3A10-mediated steroid hydroxylase activities included androstenedione 16 alpha-hydroxylation, progesterone 16 alpha- and 21-hydroxylation, and the formation of several unidentified products. CYP 3A10 exhibited similar Vmax. values for the 6 beta-hydroxylation of androstenedione and lithocholic acid (132 and 164 pmol/min per mg respectively), but metabolized the bile acid with a 3-fold lower Km (25 microM, as against 75 microM for androstenedione). Together, these studies establish that the substrate specificity of the bile acid hydroxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabolism of two classes of endogenous P450 substrates:steroid hormones and bile acids.

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Background Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings on the progressive changes in gene expression induced by MAA in a cultured Leydig cell model may help elucidate signaling pathways that lead to the testicular pathophysiological responses induced by MAA exposure and may identify useful biomarkers of MAA toxicity.

The primary focus of chemoprevention research is the prevention of cancer using pharmacological, biological, and nutritional interventions. Chemotherapeutic approaches that have been used successfully for both the prevention and treatment of a number of human malignancies have arisen from the identification of specific agents and appropriate molecular targets. Although drug-metabolizing enzymes have historically been targeted in attempts to block the initial, genotoxic events associated with the carcinogenic process, emerging evidence supports the idea that manipulating drug-metabolizing enzymes may also be an effective strategy to be used for treating tumor progression, invasion, and, perhaps, metastasis. This report summarizes a symposium that presents some recent progress in this area. One area of emphasis is the development of a CYP17 inhibitor for treatment of prostate cancer that may also have androgen-independent anticancer activity at higher concentrations. A second focus is the use of a mouse model to investigate the effects of aryl hydrocarbon receptor and Cyp1b1 status and chemopreventative agents on transplacental cancer. A third area of focus is the phytochemical manipulation of not only cytochrome P450 (P450) enzymes but also phase II inflammatory and antioxidant enzymes via the nuclear factor-erythroid 2-related factor 2 pathway to block tumor progression. A final highlight is the use of prodrugs activated by P450 enzymes to halt tumor growth and considerations of dosing schedule and targeted delivery of the P450 transgene to tumor tissue. In addition to highlighting recent successes in these areas, limitations and areas that should be targeted for further investigation are discussed.

The hydroxylation of cholesterol, bile acids, and steroid hormones by liver cytochrome P450 (CYP) enzymes proceeds with a high degree of regiospecificity, and contributes to both biosynthetic and catabolic pathways of sterol metabolism. CYP 7-catalyzed cholesterol 7α-hydroxylation, a key control point of bile acid biosynthesis, is regulated at a pretranslational step, probably transcription initiation, by multiple factors, including liver bile acid and cholesterol levels, thyroid hormone status, and diurnal rhythm. Hydrophobic bile acids, such as lithocholic acid, are converted to less cholestatic derivatives by 6β-hydroxylation carried out by CYP 3A P450s, which also catalyze steroid hormone 6β-hydroxylation reactions. Complex, gender-dependent developmental patterns characterize the expression of steroid 5α-reductase and several rat liver steroid hydroxylase CYPs. Multiple pituitary-dependent factors regulate the expression of these enzymes; of greatest importance are the gonadal steroids and the sex-dependent secretory patterns of growth hormone (GH) that they impart. The continuous presence of GH in circulation, a characteristic of adult female rats, positively regulates expression of the female-specific steroid disulfate 15β-hydroxylase CYP 2C12, while expression of the male-specific steroid 16α- and 2α-hydroxylase CYP 2C11 is stimulated by the intermittent pituitary secretion of GH that occurs in adult male rats. Intermittent GH can stimulate CYP 2C11 gene expression even when the hormone presents to the hepatocyte at a non-physiological pulse amplitude, duration, and frequency, provided that an interpulse interval of no GH (obligatory recovery period) is maintained for at least 2.5 h. GH regulates the expression of the CYP 2C11 and CYP 2C12 genes at the level of transcription initiation. This process is probably mediated by sex-dependent and GH-regulated protein-DNA interactions, such as those observed in the 5'-flank of the CYP 2C12 gene. Thyroid hormone is a second major regulator of liver steroid hydroxylase P450 activity. It regulates these enzymes directly, at a pretranslational step, and indirectly, through its stimulation of pituitary GH secretion and by its positive effects on the expression of the flavoenzyme NADPH-P450 reductase, which catalyzes electron transfer that is obligatory for all microsomal steroid hydroxylation reactions.

Patients with testicular cancer treated with cisplatin can undergo feminization that is understood poorly. Rat model studies recently showed that cisplatin can feminize in part the profile of hepatic steroid-metabolizing enzymes and circulating hormone levels. This study was undertaken to determine whether cisplatin similarly might contribute to the perturbations in gonadotropin or steroid hormone levels that can occur in patients undergoing cisplatin-based treatment for testicular cancer. Analysis of serum free testosterone, total testosterone, and androstenedione levels revealed that these hormones were not altered significantly in patients during a 38-week period of cisplatin-based treatment and follow-up. Estradiol levels were elevated before chemotherapy and were reduced to normal levels during treatment. This reduction was attributed to the cytotoxic effect of chemotherapy on the tumors and the resultant reduction in serum chorionic gonadotropin levels. Serum dihydrotestosterone (DHT) levels were normal before chemotherapy but progressively became elevated during treatment with cisplatin in five of ten patients examined. The rise in DHT may relate to the previously described increase in hepatic androgen 5 alpha-reductase activity in cisplatin-treated rats. Levels of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone (FSH) were normal before cisplatin-based treatment was administered; however, FSH was elevated selectively during chemotherapy. This selective induction of FSH may reflect an effect of cisplatin on the hypothalamic secretion of gonadotropin-releasing hormone. Taken together, these findings suggest that cisplatin contributes to the perturbation of steroid and peptide hormone levels in patients with testicular cancer and perhaps in others undergoing cisplatin-based chemotherapy.

Hybridomas obtained by the fusion of spleen cells from rat cytochrome b 5 -immunized mice with mouse myeloma cells produced five groups of monoclonal antibodies (MAbs) with three mouse immunoglobulin subtypes: IgG1, IgG2b and IgM. All of the MAbs bound strongly to rat cytochrome b 5 as measured by radioimmunoassay (RIA). Four clones of MAbs were also strongly immunoreactive with cytochrome b 5 when tested by Western blotting, but only one of the MAbs (1-39-2) weakly immunoprecipitated cytochrome b 5 in an Ouchterlony double-immunodiffusion test. Two of the MAbs partially inhibited cytochrome b 5 -mediated NADH cytochrome c reduction catalyzed by liver microsomes (24–36%). Expression of immunodetectable cytochrome b 5 was highest in the liver, next highest in the kidney, and quite low in the other tissues examined with MAb 1-17-1 by Western blotting. This MAb recognized homologous cytochrome b 5 of human liver microsomes and in homogenates of TK − cells infected with recombinant vaccinia virus encoding human cytochrome b 5 . These MAbs to cytochrome b 5 will be useful for the identification, quantification, and purification of cytochrome b 5 from animal and human tissues, and for understanding its role in cytochrome P450 catalyzed drug metabolism and carcinogen activation with respect to tissue, organ and individual differences.

The induction of liver cytochrome P450 4A-catalyzed fatty acid omega-hydroxylase activity by clofibrate and other peroxisome proliferators has been proposed to be causally linked to the ensuing proliferation of peroxisomes in rat liver. Since female rats are less responsive than males to peroxisome proliferation induced by clofibrate, the influence of gender and hormonal status on the basal and clofibrate-inducible expression of the 4A P450s was examined. Northern blot analysis using gene-specific oligonucleotide probes revealed that in the liver, P450 4A1 and 4A3 mRNAs are induced to a much greater extent in male as compared to female rats following clofibrate treatment, whereas P450 4A2 mRNA is altogether absent from female rat liver. Male-specific expression of P450 4A2 mRNA was also observed in kidney. Western blot analysis indicated that a similar sex dependence characterizes both the basal expression and the clofibrate inducibility of the corresponding P450 4A proteins. This suggests that the lower responsiveness of female rats to clofibrate-induced peroxisome proliferation may reflect the lower inducibility of the P450 4A fatty acid hydroxylase enzymes in this sex. Investigation of the contribution of pituitary-dependent hormones to the male-specific expression of 4A2 revealed that this P450 mRNA is fully suppressed in liver following exposure to the continuous plasma growth hormone profile that characterizes adult female rats; in this and other regards liver P450 4A2 is regulated in a manner that is similar, but not identical to, P450 3A2, a male-specific testosterone 6 beta-hydroxylase. In contrast, kidney 4A2 expression, although also male-specific, was not suppressed by continuous growth hormone treatment, but was regulated by pathways that, in part, involve testosterone as a positive regulator. The male-specific expression of liver and kidney P450 4A2 is thus under the control of distinct pituitary-dependent hormones acting in a tissue-specific manner.

Growth hormone (GH) differentially regulates the expression of several male-specific and female-specific liver cytochrome P450 mRNAs as a function of its sex-dependent ultradian secretory pattern. Pulsatile GH release stimulates expression of the male-specific P450 2C11, while a continuous GH secretion pattern suppresses expression of 2C11 and stimulates the expression of the female-specific P450 2C12. To help define the level at which GH regulates the expression of 2C11 and 2C12 mRNA, liver nuclear RNA samples isolated from rats differing in GH status were analyzed for 2C11 and 2C12 hnRNAs by hybridization to 2C11 and 2C12 gene-specific exonic oligonucleotide probes, as well as exon/intron junction probes. The 2C11 and 2C12 hnRNAs were found to be responsive to circulating GH profiles in a manner indistinguishable from the corresponding mature, cytoplasmic mRNAs, with no 2C12 mRNA precursors found in untreated male or hypophysectomized female liver nuclei, and no 2C11 mRNA precursors in untreated female or hypophysectomized male liver nuclei. Thus, transport of 2C11 and 2C12 RNA to the cytoplasm and cytoplasmic mRNA stability are unlikely to be important GH-regulated control points for sex-specific P450 RNA expression. Run-on transcription analysis further established that GH regulates the sex-specific expression of the 2C11 and 2C12 genes at the level of transcript initiation. Transcription was also shown to be the major step for regulation of the male-specific P450 2A2 RNA, whose expression, unlike 2C11, is not obligatorily dependent on pulsatile GH release. In vitro footprinting analysis of 2C11 and 2C12 promoter fragments incubated with liver nuclear proteins isolated from rats differing in GH status revealed several sex- and GH-dependent differences in DNase cleavage patterns ("hypersensitivity sites"), demonstrating that GH can regulate specific protein-DNA interactions in the 5'-flanking sequences of these two genes. In vitro transcription assays driven by 2C11 and 2C12 5'-flanking DNA sequences fused to TATAA box-G-less cassette template constructs did not, however, faithfully mimic the sex-specific transcription of the 2C11 and 2C12 genes, indicating that additional cis-elements or trans-acting factors may be required to achieve the transcriptional regulation of these genes that occurs in vivo.

Sex differences in pharmacokinetics and pharmacodynamics characterize many drugs and contribute to individual differences in drug efficacy and toxicity. Sex-based differences in drug metabolism are the primary cause of sex-dependent pharmacokinetics and reflect underlying sex differences in the expression of hepatic enzymes active in the metabolism of drugs, steroids, fatty acids and environmental chemicals, including cytochromes P450 (P450s), sulfotransferases, glutathione transferases, and UDP-glucuronosyltransferases. Studies in the rat and mouse liver models have identified more than 1000 genes whose expression is sex-dependent; together, these genes impart substantial sexual dimorphism to liver metabolic function and pathophysiology. Sex differences in drug metabolism and pharmacokinetics also occur in humans and are due in part to the female-predominant expression of CYP3A4, the most important P450 catalyst of drug metabolism in human liver. The sexually dimorphic expression of P450s and other liver-expressed genes is regulated by the temporal pattern of plasma growth hormone (GH) release by the pituitary gland, which shows significant sex differences. These differences are most pronounced in rats and mice, where plasma GH profiles are highly pulsatile (intermittent) in male animals versus more frequent (nearly continuous) in female animals. This review discusses key features of the cell signaling and molecular regulatory mechanisms by which these sex-dependent plasma GH patterns impart sex specificity to the liver. Moreover, the essential role proposed for the GH-activated transcription factor signal transducer and activator of transcription (STAT) 5b, and for hepatic nuclear factor (HNF) 4α, as mediators of the sex-dependent effects of GH on the liver, is evaluated. Together, these studies of the cellular, molecular, and gene regulatory mechanisms that underlie sex-based differences in liver gene expression have provided novel insights into the physiological regulation of both xenobiotic and endobiotic metabolism.

The hormonal regulation of human CYP2C18 and CYP2C19, which are expressed in a male-specific manner in liver and kidney in a mouse transgenic model, was examined. The influence of prepubertal castration in male mice and testosterone treatment of female mice was investigated, as was the effect of continuous administration of growth hormone (GH) to transgenic males. Prepubertal castration of transgenic male mice suppressed the expression of CYP2C18 and CYP2C19 in liver and kidney to female levels, whereas expression was increased for the endogenous female-specific mouse hepatic genes Cyp2c37, Cyp2c38, Cyp2c39, and Cyp2c40. Testosterone treatment of female mice increased CYP2C18 and CYP2C19 expression in kidney, and to a lesser extent in liver, but was without effect in brain or small intestine, where gene expression was not gender-dependent. Continuous GH treatment of transgenic males for 7 days suppressed hepatic expression of CYP2C19 (>90% decrease) and CYP2C18 (∼50% decrease) but had minimal effect on the expression of these genes in kidney, brain, or small intestine. Under these conditions, continuous GH induced all four female-specific mouse liver Cyp2c genes in males to normal female levels. These studies indicate that the human CYP2C18 and CYP2C19 genes contain regulatory elements that respond to the endogenous mouse hormonal profiles, with androgen being the primary regulator of male-specific expression in kidney, whereas the androgen-dependent pituitary GH secretory pattern is the primary regulator of male-specific expression in liver in a manner that is similar to the regulation of the endogenous gender-specific hepatic genes.

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.

Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.

Neonatal male rats were treated with monosodium glutamate (MSG) at either 0.5, 1.0, 2.0, 3.0 or 4.0 mg/g body weight on alternate days during the first 9 days of life. As adults, rats were catheterized to obtain unstressed, serial blood samples for the determination of ultradian patterns of circulating growth hormone. In addition, the levels of drug-metabolizing enzymes (i.e. hexobarbital hydroxylase, cytochromes P450 and b 5 , NADPH-cytochrome P450 reductase and ethoxyresorufin O -deethylase as well as sex-dependent forms of cytochrome P450 [i.e. male-dependent cytochromes P450 2c (IIC11), 2a (IIIA2) and RLM2 (IIA2) and female-dependent cytochromes P450 2d (IIC12) and 3 (IIA1)] and/or their catalytic activities were measured in the hepatic microsomes of the treated rats. The results demonstrated a dose-dependent, graded response to MSG treatment. As the dose of MSG increased from 0.5 to 4.0 mg, there was a concurrent decline in the amplitudes of the characteristically masculine, episodic bursts of growth hormone, until at the highest dose (4mg), the pulses were no longer detectable. Associated with this dose-dependent alteration in the ultradian pattern of growth hormone secretion was a measurable change in the activities of the sex-dependent hepatic enzymes. As the pulse heights of the hormone declined to 10–20% of their normal amplitudes, the levels of the male-dependent enzymes (i.e. the drug-metabolizing enzymes, as well as the male forms of cytochrome P450 and their specific steroid hydroxylases) were maintained, and in some cases, exceeded the levels normally found in males. However, as the hormone pulse heights declined, there appeared an accompanying increase in the activities of some of the female-dependent enzymes. Finally, with the loss of all detectable levels of circulating growth hormone, the normal masculine profile of hepatic enzymes was reversed to an apparently normal (with the exception of cytochrome P450 2d) feminine profile. Summarizing, the results indicate that (1) neonatal administration of MSG can produce dose-dependent, graded, long-term developmental defects in the ultradian rhythm of circulating growth hormone and associated sex-dependent hepatic enzymes, and (2) while the male-dependent hepatic enzymes can be maintained at normal or even higher levels in the face of an up to 90% reduction in the pulse heights of plasma growth hormone, the activities of the female-dependent enzymes may begin to increase.

We provide here a list of 154 P450 genes and seven putative pseudogenes that have been characterized as of October 20, 1990. These genes have been described in a total of 23 eukaryotes (including nine mammalian and one plant species) and six prokaryotes. Of 27 gene families so far described, 10 exist in all mammals. These 10 families comprise 18 subfamilies, of which 16 and 14 have been mapped in the human and mouse genomes, respectively; to date, each subfamily appears to represent a cluster of tightly linked genes. We propose here a modest revision of the initially proposed (Nebert et al., DNA 6, 1-11, 1987) and updated (Nebert et al., DNA 8, 1-13, 1989) nomenclature system based on evolution of the superfamily. For the gene we recommend that the italicized root symbol CYP for human (Cyp for mouse), representing cytochrome P450, be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. We suggest that the human nomenclature system be used for other species. This system is consistent with our earlier proposed nomenclature for P450 of all eukaryotes and prokaryotes, except that we are discouraging the future use of cumbersome Roman numerals.

Studies carried out in hypophysectomized adult rats have demonstrated that both thyroid hormone and GH can suppress hepatic expression of the steroid 6 beta-hydroxylase P450 2a (IIIA2). The present study further characterizes the influence of thyroid hormone on the expression of P450 2a and two other male-specific hepatic P450s, a steroid 2 alpha/16 alpha-hydroxylase, designated P450 2c (IIC11), and a steroid 15 alpha-hydroxylase, designated P450 RLM2 (IIA2). These studies were carried out in rats rendered hypothyroid by treatment with methimazole, which allows for the nonsurgical depletion of circulating T4, and in hypophysectomized rats. Hypothyroidism led to an increase in hepatic P450 2a (IIIA2) protein and mRNA in both male and female rats that was fully reversed by T4 replacement. In contrast, hypothyroidism decreased by 70-80% the expression of P450 2c (IIC11) activity and mRNA, but did not significantly alter the expression of P450 RLM2 (IIA2). The decrease in P450 2c (IIC11) was not reversed by T4 replacement, suggesting that it is a consequence of the loss of plasma GH pulses that occurs secondary to hypothyroidism. In agreement with these findings, T4 given to hypophysectomized rats partially suppressed the expression of P450 2a (IIIA2) mRNA, but not P450 2c (IIC11) or P450 RLM2 (IIA2) mRNA. A more complete suppression of P450 2a (IIIA2) mRNA as well as P450 2c (IIC11) mRNA was achieved when the hypophysectomized rats were treated with T3 at a supraphysiological, receptor-saturating dose. Although GH administered to intact male rats by continuous infusion fully suppressed all three male-specific P450 proteins and their mRNAs, the same treatment given to hypothyroid rats was only partially suppressive in the case of P450 2a (IIIA2) and P450 RLM2 (IIA2), unless combined with T4. In the case of P450 2c (IIC11), substantial suppression of the residual P450 present in hypothyroid rats was achieved by treatment with GH alone, despite persistent thyroid hormone deficiency. These studies demonstrate that while thyroid hormone is a negative regulator of P450 2a (IIIA2) expression and is required for the full suppression of that P450 and P450 RLM2 (IIA2) by the continuous plasma GH profiles associated with adult female rats, the suppression of P450 2c (IIC11) by continuous plasma GH is largely independent of the presence of thyroid hormone.

Preclinical and clinical studies of CYP gene-directed enzyme prodrug therapy have been focused on anticancer prodrugs activated by CYP2B enzymes, which have low endogenous expression in human liver; however, the gene therapeutic potential of CYP3A enzymes, which are highly expressed in human liver, remains unknown. This study investigated methoxymorpholinyl doxorubicin (MMDX; nemorubicin), a novel CYP3A-activated anticancer prodrug. Retroviral transfer of CYP3A4 increased 9L gliosarcoma cell chemosensitivity to MMDX 120-fold (IC(50)=0.2 nM in 9L/3A4 cells). In CHO cells, overexpression of P450 reductase in combination with CYP3A4 enhanced chemosensitivity to MMDX, and to ifosfamide, another CYP3A4 prodrug, 11- to 23-fold compared with CYP3A4 expression alone. CYP3A4 expression and MMDX chemosensitivity were increased in human lung (A549) and brain (U251) tumor cells infected with replication-defective adenovirus encoding CYP3A4. Coinfection with Onyx-017, a replication-conditional adenovirus that coamplifies and coreplicates the Adeno-3A4 virus, led to large increases in CYP3A4 RNA but only modest increases in CYP3A4 protein and activity. MMDX induced remarkable growth delay of 9L/3A4 tumors, but not the P450-deficient parental 9L tumors, in immunodeficient mice administered low-dose MMDX either intravenous or by direct intratumoral (i.t.) injection (60 microg kg(-1), every 7 days x 3). Notably, the i.t. route was substantially less toxic to the mouse host. No antitumor activity was observed with intraperitoneal MMDX treatment, suggesting a substantial hepatic first pass effect, with activated MMDX metabolites formed in the liver having poor access to the tumor site. These studies demonstrate that human CYP3A4 has strong potential for MMDX prodrug-activation therapy and suggest that endogenous tumor cell expression of CYP3A4, and not hepatic CYP3A4 activity, is a key determinant of responsiveness to MMDX therapy in cancer patients in vivo.

Pituitary GH-secretory profiles are sex dependent and regulate the sexually dimorphic expression of a large number of genes in the liver. The slow response of many sex-specific liver genes to changes in plasma GH status suggests that GH acts in the liver via both direct and indirect mechanisms organized in a hierarchical regulatory network. Presently, genome-wide liver transcription profiling was conducted to elucidate the global impact of pituitary hormone ablation on the sex specificity of rat liver gene expression and to identify sex-specific genes that respond rapidly to GH as candidates for direct targets of GH action. Hypophysectomy abolished the sex specificity of approximately 90% of 1032 sex-dependent genes, consistent with the dominant role of pituitary GH in regulating liver sexual dimorphism. Two major classes of sex-specific genes were identified: genes that were down-regulated after hypophysectomy and may be subject to positive GH regulation (461 class I genes), and genes that were up-regulated after hypophysectomy and may be subject to negative GH regulation (224 class II genes). Fifty class I sex-specific genes were induced, and 38 class II sex-specific genes were suppressed within 90 min of a physiological GH pulse, suggesting they are primary GH response genes. A further 71 sex-specific genes responded after a second GH treatment and may correspond to secondary response genes. Twenty four DNA-binding proteins were identified as early GH response genes, of which 15 were induced and nine were suppressed by GH. Five of these 24 genes displayed sex-specific expression, consistent with a hierarchical transcriptional network controlling sex-specific liver gene expression. Class II male-specific genes, such as Cyp2a2 and Cyp2c13, were down-regulated within 30 min of GH pulse treatment, as determined by heterogeneous nuclear RNA analysis, suggesting that transcription of these genes is restricted to the GH-free interpulse period in adult male rat liver. We conclude that GH acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release; this release is intermittent in male rats and nearly continuous in females. Comparisons of liver nuclear protein tyrosine phosphorylation in male and female rats have led to the discovery that the liver transcription factor STAT5b is tyrosine phosphorylated in male but not female rats in response to GH pulses. Intermittent plasma GH pulses trigger a rapid and repeated tyrosine phosphorylation and nuclear translocation of liver STAT5b in intact male rats, while the more continuous pattern of GH exposure down-regulates the STAT5b signalling pathway in female rat liver. The central importance of STAT5b for the physiological effects of GH pulses has been verified using a mouse gene knockout model. STAT5b gene disruption leads to a major loss of multiple sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression are decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products are increased in males to near female levels. STAT5b is thus a liver-expressed, latent cytoplasmic transcription factor that undergoes repeated tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation, and is a key intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene transcription. Other studies indicate, however, that STAT5a and STAT5b are both required for constitutive expression in female, but not male liver, of certain GH-regulated CYP enzymes. GH activation of both STAT5 proteins, which in turn form distinct homodimeric and heterodimeric DNA-binding complexes, is thus an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.

Endocrine disruptive compounds (EDC) alter hormone-stimulated, nuclear receptor-dependent physiological and developmental processes by a variety of mechanisms. One recently identified mode of endocrine disruption is through hormone sensitization, where the EDC modulates intracellular signaling pathways that control nuclear receptor function, thereby regulating receptor transcriptional activity indirectly. Methoxyacetic acid (MAA), the primary, active metabolite of the industrial solvent ethylene glycol monomethyl ether and a testicular toxicant, belongs to this EDC class. Modulation of nuclear receptor activity by MAA could contribute to the testicular toxicity associated with MAA exposure. In the present study, we evaluated the impact of MAA on the transcriptional activity of several nuclear receptors including the androgen receptor (AR), which plays a pivotal role in the development and maturation of spermatocytes. AR transcriptional activity is shown to be increased by MAA through a tyrosine kinase signaling pathway that involves PI3-kinase. In a combinatorial setting with AR antagonists, MAA potentiated the AR response without significantly altering the EC(50) for androgen responsiveness, partially alleviating the antagonistic effect of the anti-androgens. Finally, MAA treatment of TM3 mouse testicular Leydig cells markedly increased the expression of Cyp17a1 and Shbg while suppressing Igfbp3 expression by ~90%. Deregulation of these genes may alter androgen synthesis and action in a manner that contributes to MAA-induced testicular toxicity.

Hepatocyte nuclear factor (HNF)-4alpha is a liver-enriched transcription factor that regulates numerous liver-expressed genes including several sex-specific cytochrome P450 genes. Presently, a liver-specific HNF4alpha-deficient mouse model was used to characterize the impact of liver HNF4alpha deficiency on a global scale using 41,174 feature microarrays. A total of 4994 HNF4alpha-dependent genes were identified, of which about 1000 fewer genes responded to the loss of HNF4alpha in female liver as compared with male liver. Sex differences in the impact of liver HNF4alpha deficiency were even more dramatic when genes showing sex-specific expression were examined. Thus, 372 of the 646 sex-specific genes characterized by a dependence on HNF4alpha responded to the loss of HNF4alpha in males only, as compared with only 61 genes that responded in females only. Moreover, in male liver, 78% of 508 male-specific genes were down-regulated and 42% of 356 female-specific genes were up-regulated in response to the loss of HNF4alpha, with sex specificity lost for 90% of sex-specific genes. This response to HNF4alpha deficiency is similar to the response of male mice deficient in the GH-activated transcription factor signal transducer and activator of transcription 5b (STAT5b), where 90% of male-specific genes were down-regulated and 61% of female-specific genes were up-regulated, suggesting these two factors cooperatively regulate liver sex specificity by mechanisms that are primarily active in males. Finally, 203 of 648 genes previously shown to bind HNF4alpha near the transcription start site in mouse hepatocytes were affected by HNF4alpha deficiency in mouse liver, with the HNF4alpha-bound gene set showing a 5-fold enrichment for genes positively regulated by HNF4alpha. Thus, a substantial fraction of the HNF4alpha-dependent genes reported here are likely to be direct targets of HNF4alpha.

The promising but still limited efficacy of angiogenesis inhibitors as monotherapies for cancer treatment indicates a need to integrate these agents into existing therapeutic regimens. Presently, we investigate the antitumor activity of the small-molecule angiogenesis inhibitor axitinib (AG-013736) and its potential for combination with metronomic cyclophosphamide. Axitinib significantly inhibited angiogenesis in rat 9L tumors grown s.c. in scid mice but only moderately delayed tumor growth. Combination of axitinib with metronomic cyclophosphamide fully blocked 9L tumor growth on initiation of drug treatment. In contrast, metronomic cyclophosphamide alone required multiple treatment cycles to halt tumor growth. However, in contrast to the substantial tumor regression that is ultimately induced by metronomic cyclophosphamide, the axitinib/cyclophosphamide combination was tumor growth static. Axitinib did not inhibit hepatic activation of cyclophosphamide or export of its activated metabolite, 4-hydroxy-cyclophosphamide (4-OH-CPA), to extrahepatic tissues; rather, axitinib selectively decreased 9L tumor uptake of 4-OH-CPA by 30% to 40%. The reduced tumor penetration of 4-OH-CPA was associated with a decrease in cyclophosphamide-induced tumor cell apoptosis and a block in the induction of the endogenous angiogenesis inhibitor thrombospondin-1 in tumor-associated host cells, which may contribute to the absence of tumor regression with the axitinib/cyclophosphamide combination. Finally, axitinib transiently increased 9L tumor cell apoptosis, indicating that its effects are not limited to the endothelial cell population. These findings highlight the multiple effects that may characterize antiangiogenic agent/metronomic chemotherapy combinations and suggest that careful optimization of drug scheduling and dosages will be required to maximize antitumor responses. [Mol Cancer Ther 2008;7(1):79–89]

The sexually differentiated microsomal enzyme steroid 5 alpha-reductase (NADPH: delta 4-3-oxosteroid 5 alpha-oxido-reductase, EC 1.3.99.5) catalyzes the NADPH-dependent conversion of testosterone to 5 alpha-dihydrotestosterone, a more potent androgen. In rat liver, this enzyme is expressed at a 10-fold higher level in adult females as compared to adult males. The pituitary regulation of this enzyme and its mRNA was studied in untreated and hypophysectomized rats and in rats rendered hypothyroid by treatment with the antithyroid drug methimazole. Hepatic 5 alpha-reductase activity was elevated 8-fold, to 85% of adult female levels, in adult male rats given growth hormone by continuous infusion. This same treatment was only partially effective in restoring 5 alpha-reductase in rats depleted of endogenous growth hormone by hypophysectomy, indicating that other pituitary-dependent factors contribute to the elevation observed in the inact animals. Further analysis revealed that thyroxine, but not adrenocorticotropic hormone (ACTH) or chorionic gonadotropin, could elevate 5 alpha-reductase activity and mRNA when given to the hypophysectomized rats and that this effect was enhanced by the presence of growth hormone. This thyroid hormone dependence was confirmed by the decrease in hepatic 5 alpha-reductase expression in hypothyroid rats and by its substantial restoration following thyroxine replacement. Thyroxine also stimulated expression of another female-predominant hepatic mRNA, encoding the steroid 16 alpha-hydroxylase cytochrome P-450f (IIC7), in a manner that was independent of the stimulatory effect of growth hormone on this transcript. In contrast, thyroid hormone did not significantly affect protein or mRNA levels of the growth hormone-stimulated, female-specific steroid sulfate 15 beta-hydroxylase P-450 2d (IIC12). These findings establish that thyroid hormones act at a pretranslational level to modulate the expression of some, but not all, growth hormone-stimulated hepatic mRNAs and demonstrate that both thyroxine and growth hormone can independently contribute to the sex-dependent expression of hepatic enzymes of steroid metabolism.

Cholesterol 7 alpha-hydroxylase (P-450 Ch7 alpha) catalyzes the first and rate-limiting step in the hepatic conversion of cholesterol to bile acids. P-450 Ch7 alpha activity in rat liver is regulated at three independent levels: (a) feedback inhibition by bile acids (long term regulation); (b) midterm regulation through the diurnal cycle; (c) short term modulation by hormones and dietary factors. P-450 Ch7 alpha was purified to apparent homogeneity and in active form (turnover number = 10-15 min-1 P-450(-1)) from cholestyramine-fed female rats, and rabbit anti-P-450 Ch7 alpha polyclonal antibodies were then prepared. Liver microsomes were isolated from rats fed normal diet or diet containing the bile acid sequestrant cholestyramine and were then killed at either the apex (midnight) or nadir (noon) of the diurnal rhythm of P-450 Ch7 alpha activity. Direct comparison of microsomal P-450 Ch7 alpha enzyme activity levels with P-450 Ch7 alpha protein (Western blotting) and mRNA levels (Northern and slot blots) revealed that the 2.5-3-fold induction of P-450 Ch7 alpha activity with cholestyramine feeding can be fully accounted for by an increase in P-450 Ch7 alpha protein and mRNA. Turnover numbers of 7-9 nmol of 7 alpha-hydroxycholesterol/min/nmol of microsomal P-450 Ch7 alpha were observed for both induced and uninduced animals. Similarly, the postmidnight decrease in enzyme activity could be generally accounted for by a decrease in P-450 Ch7 alpha protein and mRNA, suggesting that these species have relatively short half-lives. The short term regulation of P-450 Ch7 alpha was examined following treatment with the cholesterol precursor mevalonic acid. A 2.5-fold increase in hepatic microsomal P-450 Ch7 alpha activity occurred within 150 min and was accompanied by a significant elevation of P-450 Ch7 alpha mRNA (up to 3-6-fold increase). These findings establish that hepatic cholesterol 7 alpha-hydroxylase activity is regulated in response to long term, midterm, and short term control factors primarily at a pretranslational level and that this regulation is of greater importance than proposed mechanisms based on allosteric effects of bile acids on P-450 Ch7 alpha protein, changes in cholesterol availability, or reversible phosphorylation of a putative P-450 Ch7 alpha phosphoprotein.

The influence of lipophilicity and carboxyl group content on the ability of methotrexate (MTX) derivatives to undergo 7-hydroxylation in vitro by partly purified rabbit hepatic aldehyde oxidase was examined. Addition of two to four γ-glutamyl residues to the MTX molecule caused a progressive decrease in the rate of hydroxylation associated mainly with a decrease in Vmax rather than an increase in Km. These results suggest that the number of carboxyl groups in the side chain has a relatively small effect on affinity for the enzyme active site, but hinders the formation of product. The catalytic efficiency of hydroxylation of MTX tetraglutamate, estimated from V max K m ratios, was 36-fold lower than that of the monoglutamate. In contrast, when the number of carboxyl groups was decreased to one, as in 4-amino-4-deoxy-N10-methylpteroic acid, N α -(4- amino -4- deoxy -N 10 - methylpteroyl )- l - lysine , and γ-t-butyl-3′-chloromethotexate, enhanced catalytic efficiency was observed, involving both a decrease in Km and an increase in Vmax. The catalytic efficiency of hydroxylation of these three substrates was 88-, 360-and 2100-fold higher than that of MTX. γ-t-Butyl-3′-chloromethotrexate was a better substrate than γ-t-butyl-MTX, demonstrating the strong contribution of a lipophilic Cl atom on the phenyl ring. N α -(4- Amino -4- deoxypteroyl )-N δ - hemiphthaloyl - l - ornithine , with two carboxyl groups, showed substrate activity similar to that of MTX. The γ-t-butyl esters of MTX, 3′-chloromethotrexate, and 3′,5′-dichloromethotrexate were compared with the parent acids as inhibitors of the growth of cultured human leukemic lymphoblasts (CEM cells) and an MTX-resistant subline (CEM/MTX) defective in MTX transport and polyglutamylation. Although the esters were less effective than the acids against CEM cells except at high concentrations, they were more effective against CEM/MTX cells. This “collateral sensitivity” of CEM/MTX cells to lipophilic MTX esters is consistent with a decreased ability to take up and utilize reduced folates from the culture medium.