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Cyclophosphamide (CPA), a widely used oxazaphosphorine anti-cancer prodrug, is inactive until it is metabolized by cytochrome P450 to yield phosphoramide mustard and acrolein, which alkylate DNA and proteins, respectively. Tumor cells transduced with the human cytochrome P450 gene CYP2B6 are greatly sensitized to CPA, however, the pathway of CPA-induced cell death is unknown. The present study investigates the cytotoxic events induced by CPA in 9L gliosarcoma cells retrovirally transduced with CYP2B6, or induced in wild-type 9L cells treated with mafosfamide (MFA) or 4-hydroperoxyifosfamide (4OOH-IFA), chemically activated forms of CPA and its isomer ifosfamide. CPA and MFA were both shown to effect tumor cell death by stimulating apoptosis, as evidenced by the induction of plasma membrane blebbing, DNA fragmentation, and cleavage of the caspase 3 and caspase 7 substrate poly(ADP-ribose) polymerase (PARP) in drug-treated cells. Caspase 9 was identified as the regulatory upstream caspase activated in 9L cells treated with CPA, MFA, or 4OOH-IFA, implicating the mitochondrial apoptotic pathway in oxazaphosphorine-induced tumor cell death. Correspondingly, expression of the mitochondrial proapoptotic factor Bax enhanced caspase 9 activation, plasma membrane blebbing, and drug-induced cytotoxicity. Conversely, overexpression of the mitochondrial antiapoptotic factor Bcl-2 blocked caspase 9 activation, leading to an inhibition of drug-induced plasma membrane permeability and blebbing, terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity, PARP cleavage, Annexin V positivity, and drug-induced cell death. Although Bcl-2 thus blocked the cytotoxic effects of activated CPA, it did not inhibit the drug's cytostatic effects. CPA induced S-phase cell cycle arrest followed by conversion to an apoptotic pre-G1 state in wild-type 9L cells; by contrast, Bcl-2-expressing 9L cells accumulated in G2/M in response to CPA treatment. Intratumoral expression of Bcl-2 and related family members, including both apoptotic and antiapoptotic factors, is thus an important determinant of the responsiveness of tumor cells to CPA and ifosfamide, both in the context of conventional chemotherapy and in patients sensitized to these oxazaphosphorine drugs by the use of cytochrome P450-based gene therapy.

Studies were carried out to elucidate the mechanism whereby thyroid hormone (T3) induces NADPH:cytochrome P450 oxidoreductase (P450R) mRNA in rat liver in vivo. Northern blot analysis revealed that T3 treatment increases unspliced liver nuclear P450R RNA 4-fold within 8 h and that this induction precedes the induction of mature, cytoplasmic P450R RNA. Unspliced nuclear P450R RNA was suppressed below basal levels 24 h after T3 treatment, despite the continued presence of elevated circulating T3 levels. To determine whether the T3-stimulated increase in nuclear P450R RNA reflects an increase in P450R transcription initiation, nuclear run-on transcription assays were carried out. T3 induced a 6- to 8-fold increase in P450R transcription rate within 12 h, sufficient to account for the observed increase in nuclear P450R precursor RNA, followed by a decrease back to basal transcription levels at 24 h, consistent with the nuclear RNA profile. Similar transcriptional increases were observed in nuclear run-on transcription studies using hybridization probes corresponding to nine different fragments of the P450R gene, spanning exon 2 to exon 16. Thus, P450R transcription initiation, not transcription elongation, is the T3-regulated event. Similar results were obtained during short (5 min) compared with long (45 min) nuclear run-on transcription assays, suggesting that changes in nuclear RNA processing or regulated degradation do not contribute to the overall RNA induction. This finding was confirmed by the ability of the RNA polymerase inhibitor actinomycin D, administered in vivo, to block T3 induction of P450R transcriptional activity. We conclude that P450R transcription, rather than nuclear RNA processing or mRNA stabilization, is the primary mechanism whereby T3 induces hepatic P450R mRNA.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) mediates the effects of foreign chemical peroxisome proliferators on liver and kidney, including the induction of peroxisomal, mitochondrial, and microsomal enzymes involved in beta-oxidation of fatty acids. However, the role of this receptor in the peroxisome proliferative effects of the endogenous steroid dehydroepiandrosterone (DHEA) is not known. DHEA-3 beta-sulfate fd(DHEA-S) is shown to induce a liver peroxisome proliferative response in rats in vivo at a dose at which DHEA is much less active, which is consistent with cultured hepatocyte studies indicating a requirement for sulfation for the activation of DHEA. Transient transfection experiments demonstrated that in contrast to the prototypic foreign chemical peroxisome proliferator pirinixic acid, DHEA-S and its 17 beta-reduced metabolite, 5-androstene-3 beta, 17 beta-diol-3 beta-sulfate, are inactive in mediating trans-activation by PPAR alpha in COS-1 cells. Two other mammalian PPAR isoforms, gamma and delta/Nucl, were also inactive with respect to DHEA-S trans-activation. To test whether PPAR alpha mediates peroxisomal gene induction by DHEA-S in intact animals, we administered DHEA-S or clofibrate to mice lacking a functional PPAR alpha gene. Both peroxisome proliferators markedly increased hepatic expression of two microsomal cytochrome P450 4A proteins as well as six mRNAs known to be associated with the peroxisomal proliferative response in wild-type mice. In contrast, neither DHEA-S nor clofibrate induced these hepatic proteins and mRNAs in PPAR alpha-deficient mice. Clofibrate-induced expression of kidney CYP4A mRNAs was also blocked in the PPAR alpha gene knockout mice. Thus, despite its unresponsiveness to steroidal peroxisome proliferators in transfection assays, PPAR alpha is obligatory for DHEA-S-stimulated hepatic peroxisomal gene induction. DHEA-S, or one of its metabolites, may thus serve as an important endogenous regulator of liver peroxisomal enzyme expression via a PPAR alpha-mediated pathway.

Growth hormone (GH) secretory patterns regulate the expression of several sex-dependent liver cytochrome P450 (CYP) genes. Studies using the hypophysectomized rat model have established that the intermittent plasma GH secretory pattern associated with adult male rats markedly stimulates liver expression of the male-specific CYP 2C11, a testosterone 2 alpha- and 16 alpha-hydroxylase, but is not required for expression of other male-specific liver enzymes, including CYP 2A2, a testosterone 15 alpha-hydroxylase, and CYP 3A2, a testosterone 6 beta-hydroxylase. In the present study, the effects of intermittent GH treatment on liver CYP expression were studied in adult rats rendered GH deficient by neonatal administration of monosodium glutamate (MSG), which depletes circulating adult GH without the global loss of other pituitary-dependent hormones that is associated with hypophysectomy. Restoration of the normal masculine circulating GH profile of six daily pulses (180-225 ng GH/ml/peak) in MSG-treated male rats by the use of an external pumping apparatus led to a substantial (30-50%) restoration of normal male levels of CYP 2A2 and CYP 3A2 activity, protein, and mRNA. GH pulsation at the nonphysiological frequencies of two or four times per day was less effective unless given at a dose that resulted in supraphysiological plasma GH levels. Although intermittent GH treatment can induce male-specific P450 expression in hypophysectomized female rats, the same hormone treatment did not stimulate CYP 2A2 or CYP 3A2 expression in MSG-treated female rats. Liver GH receptor mRNA levels at adulthood were not significantly altered by neonatal MSG treatment, suggesting that the unresponsiveness of MSG-treated females and the previously reported low responsiveness of MSG-treated males to GH-induced CYP 2C11 expression are not due to the absence of GH receptor. Moreover, normal liver IGF-1 mRNA levels were expressed in the MSG-treated female rats, suggesting that the liver GH receptor is functional in these animals. The present findings establish that the adult male-specific enzymes CYP 2A2 and CYP 3A2 can be positively regulated by intermittent GH pulsation despite their GH-independent expression in hypophysectomized rats. Moreover, neonatal MSG treatment, particularly in female rats, may lead to the loss of factors other than GH that are required for full expression of the pulsatile GH-stimulated CYP 2A2, 3A2, and 2C11 genes.

Pituitary-determined hormones regulate the expression of hepatic cytochromes P-450 through processes involving both negative and positive controls. Accordingly, protein levels of several P-450 forms are elevated in rat liver following hypophysectomy [P-450 forms designated 2a (gene IIIA2), RLM2 (gene IIA2), and PB-4 (gene IIB1)], whereas protein levels of others are suppressed [e.g., P-450 2c (gene IIC11)]. In the present study, microsomal steroid hydroxylase activities associated with these same P-450 forms were found to be decreased by hypophysectomy, despite elevations in protein levels for several of them. Studies were, therefore, undertaken to determine the biochemical basis for this decrease in microsomal P-450 enzyme specific activity. In vivo treatment of hypophysectomized rats with gonadotropin, under conditions that restore heme to testis P-450, and heme reconstitution experiments carried out with liver homogenates indicated that a deficiency in P-450-associated heme is unlikely to account for the observed decreases in liver P-450 enzyme specific activity. Analysis of the flavoprotein P-450 reductase, however, revealed that the reductase protein and its associated cytochrome c reductase activity are decreased by 50 to 75% in liver microsomes isolated from hypophysectomized rats. Moreover, supplementation of isolated liver microsomes with exogenous purified P-450 reductase stimulated microsomal steroid hydroxylase activity preferentially in the hypophysectomized rats, to levels consistent with the observed changes in P-450 protein levels. Thus, a deficiency in P-450 reductase, which is a rate-limiting component for many P-450-dependent hydroxylation reactions, appears to be responsible for the decrease in steroid hydroxylase specific activity in the hypophysectomized rats. Although growth hormone, adrenocorticotropic hormone, and chorionic gonadotropin were each ineffective at restoring hepatic P-450 reductase when administered to hypophysectomized rats, substantial restoration of P-450 reductase levels could be achieved by treatment of the hypophysectomized rats with thyroxine. Thyroxine treatment of these rats also elevated the microsomal steroid hydroxylase activities associated with the individual hepatic P-450 forms to levels commensurate with their respective P-450 protein levels. These results establish that hepatic P-450 reductase is subject to hormonal controls that are distinct from those governing cytochrome P-450 expression and further demonstrate the complexity of endocrine control of hepatic steroid hormone metabolism.

The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to -536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR alpha1 was coincubated with either the retinoic X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TR beta1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.

Rat cytochrome P-450 3 (P-450 3) is a constitutive hepatic steroid hormone 7 alpha-hydroxylase which is relatively unresponsive to a number of monooxygenase-inducing agents. The present study demonstrates that a polyhalogenated aromatic hydrocarbon inducer, 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), induces P-450 3 in livers of adult male rats, and that the increase is the result of an increase in the mRNA for this enzyme. Cytochrome P-450 3 and its mRNA were increased more slowly and to a lesser extent than cytochrome P-450c (P-450c) and its mRNA, indicating that these enzymes are not regulated coordinately in liver. The maximum increase in P-450 3 and P-450 3-dependent androstenedione 7 alpha-hydroxylase activity (2- to 3-fold) occurred 7 days after administration of HCB, in contrast to the increase in P-450c (greater than 200-fold) which was maximal by 3-5 days. The rate of induction of P-450 3 mRNA was also slower [maximum increase (9-fold) at 5 days after HCB administration] than that of P-450c mRNA [maximum increase (30-fold) at 2-3 days]. Moreover, a higher dose of HCB was required to produce maximum induction of P-450 3 (50 mg/kg) than that required to produce maximum induction of P-450c (10 mg/kg). P-450 3 was not detected on Western blots of lung, kidney, or prostate microsomes isolated from control or HCB-treated rats (less than or equal to 2% of that found in livers of HCB-treated rats). Moreover, P-450 3-dependent steroid 7 alpha-hydroxylase activity was not detected in these extrahepatic tissues of control or HCB-treated rats (less than or equal to 1% of that found in the corresponding liver microsomes of untreated or HCB-treated rats). In contrast, P-450c was increased dramatically by HCB in lung, kidney, and prostate tissues, indicating differential expression of P-450c and P-450 3 in extrahepatic tissues.

Rat liver cytochrome P-450 2c (P-450 2c) is a constitutive, male-specific enzyme that oxidatively metabolizes both steroid hormones and liphophilic foreign compounds. Exposure of adult male rats to certain xenobiotics can lead to a decrease in the expression of hepatic P-450 2c. The present studies were undertaken to examine the mechanism of this decrease. Treatment of adult male rats with 3-methylcholanthrene (3-MC) or 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) (two compounds known to induce P-450c and P-450d as a consequence of their interaction with the aromatic hydrocarbon receptor) decreased hepatic content of P-450 2c and its associated steroid hormone 2 alpha- and 16 alpha-hydroxylase activities. Moreover, the present studies demonstrate that decreases in hepatic content of P-450 2c mRNA (determined by electrophoretic analysis of immunoprecipitated translational products) fully account for the effects of 3-MC and HCB on P-450 2c. HCB (50 mg/kg) produced the most striking decrease in P-450 2c and its mRNA, virtually eliminating their expression in hepatic tissue. The time course and dose-response for the decrease in P-450 2c and its mRNA differed markedly from that for induction of P-450c, indicating that the effects of HCB on the two proteins may involve different mechanisms. Additional experiments demonstrated that the sex difference in hepatic expression of P-450 2c is the result of a greater than 5-fold higher content of P-450 2c mRNA in male as compared to female rats. HCB decreased serum testosterone levels by greater than 98% at 5 days in adult male rats. However, the decrease in P-450 2c and serum testosterone levels was not accompanied by an increase in serum estradiol levels or induction of the female-specific enzyme P-450 2d. The present findings clearly establish that the decrease in P-450 2c produced by administration of HCB and 3-MC is the result of a decrease in the hepatic content of P-450 2c mRNA. These xenobiotics may decrease transcription of the mRNA for P-450 2c as a consequence of binding to the aromatic hydrocarbon receptor, or, alternatively, may interfere with the hormonal regulation of the mRNA for this male-specific P-450 enzyme.

To characterize the distribution and inducibility of drug metabolizing enzymes within different hepatic cell populations, the activities of aminopyrine N-demethylase, ethoxyresorufin O-deethylase, microsomal epoxide hydrolase and cytosolic glutathione transferase were measured in liver parenchymal, Kupffer, and endothelial cells isolated from untreated rats or rats pretreated with phenobarbital, 3-methylcholanthrene, or Aroclor 1254. Enzyme activities, measurable in all cases, were 2.3- to 5.7-fold higher in parenchymal cells than in Kupffer and endothelial cells. Phenobarbital increased aminopyrine N-demethylase, microsomal epoxide hydrolase, and cytosolic glutathione transferase activities, whereas 3-methylcholanthrene enhanced ethoxyresorufin O-deethylase, epoxide hydrolase, and glutathione transferase activities in the three cell populations. Aroclor 1254 consistently induced each of the enzyme activities in parenchymal, Kupffer, and endothelial cells. Western blot analyses revealed clear differences in the expression of proteins immunologically related to cytochrome P-450 PB-1, and glutathione transferases B and X in parenchymal cells compared with the corresponding Kupffer and endothelial cells. In contrast, only minor differences between the cell types were apparent in the expression of cytochromes P-450 PB-4, P-450 MC1a, P-450 MC1b and microsomal epoxide hydrolase. These studies establish that oxidative and postoxidative drug metabolizing enzymes are not restricted to parenchymal cells: similar but distinguishable complements of these enzymes are also found in Kupffer and endothelial cells.

Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.

Folylpolyglutamate synthetase (FPGS) catalyzes the gamma-glutamylation of both folates and folate antagonists and has been found to be essential for the survival of mammalian cells. Twelve analogs of the antifolates aminopterin (AMT) and methotrexate (MTX) having the -(CH2)2COOH moiety replaced by -(CH2)nX, where X = SO3H,PO3H2 or NH2, were evaluated as inhibitors of FPGS isolated from human liver. The AMT analogs were consistently found to be better inhibitors than their MTX counterparts, following the order of Km values determined for the parent antifolates as FPGS substrates. For the amino and phosphonate (but not for the sulfonate) compounds, inhibitory efficiencies were markedly dependent on the methylene chain length, with the most effective inhibitors having the groups -(CH2)3NH2(Ki = 0.2 microM) and -(CH2)2PO3H2 (Ki = 1.9 microM). Of those compounds exhibiting Ki values less than 200 microM, six were competitive inhibitors whereas three showed mixed inhibition (Ki' = approximately 6 Ki) when analyzed using AMT as the variable substrate. This demonstration of mixed inhibition of FPGS is consistent with the binding of inhibitor to a second site on the enzyme. Very similar Ki values (0.2-0.3 microM) were obtained for the -(CH2)3NH2 analog of AMT when using folic acid, AMT, MTX, and gamma-glutamyl-MTX as variable substrates, suggesting that the same enzymatic site on FPGS is active in the gamma-glutamylation of these four folyl derivatives. These findings serve to identify structural features which are important for inhibition of human liver FPGS and may therefore prove useful for the design of new compounds having potential as chemotherapeutic agents.