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The purpose of this study was to investigate the functional independence, intellectual performance and academic achievement of children treated for medulloblastoma at the Veterans General Hospital-Taipei. Adverse factors that it was thought would result in significant intellectual impairment were also studied. Nineteen patients with medulloblastoma located over the posterior fossa were enrolled in this study. Their mean age at tumor removal was 6.16 +/- 3.30 (range 2-14) years. Mean full intelligent quotient (FIQ) was 86.00 +/- 22.66, performance intelligent quotient (PIQ) was 85.29 +/- 21.23, and verbal intelligent quotient (VIQ) was 90.50 +/- 23.50. In 11 cases academic achievement at school was poor. Sixteen patients received radiation therapy after tumor removal. Significant negative correlations were noted between IQ and whole-brain irradiation dose (r = -0.72) and between IQ and years after radiation therapy (r = -0.63). The results indicate a need for ongoing attention and intervention to prevent, remedy or minimize deficits produced by the tumor itself and by the cancer treatment.

Role of neuropeptide Y (NPY) in noradrenergic neurotransmission has been mentioned as co-transmitter in both central and peripheral nervous system. Cerebral NPY content was changed by drugs influencing endogenous norepinephrine (NE) in rats. In an attempt to understand this mechanism, the present study was carried out using the radioimmunoassay of NPY. Values of NPY-like immunoreactivity (NPY-ir) were reduced in rats receiving the treatment of pargyline, the inhibitor of monoamine oxidase, with an elevation of catecholamine in parallel. This action was abolished by pretreatment with a mixture of phentolamine, propranolol and haloperidol at concentration sufficient to block the receptors. However, it was not influenced by treatment with haloperidol alone. Cerebrocortical NPY-ir was lowered in rats receiving an intracerebroventricular (i.c.v.) injection of methoxamine, the agonist of alpha 1-adrenoceptors. This action was prevented by prazosin via an i.c.v. injection at the dose sufficient to block alpha 1-adrenoceptors. Moreover, decrease of cerebrocortical NPY-ir by pargyline was also reversed by similar treatment of prazosin. The data obtained suggests that activation of alpha 1-adrenoceptors by endogenous NE which was increased by pargyline may lower the contents of NPY in cerebrocortex of the rat.

To characterize the dose response and time course of peripubertal testosterone imprinting of rat hepatic CYP2C11 and steroid 5 alpha-reductase and to gain further insights into the mechanism and consequences of peripubertal androgen imprinting of these enzymes, prepubertally gonadectomized female rats were injected s.c. with testosterone enanthate (5 mumol/kg/day) on days 35 to 49 (peripubertal period) or days 81 to 89 (adulthood) and then sacrificed on day 90. Androgen treatment during the peripubertal or adult period increased hepatic microsomal testosterone 2 alpha-hydroxylase activity by 4- to 5-fold and decreased steroid 5 alpha-reductase activity by 30 to 50%. By comparison, androgen administration during both periods completely masculinized these two enzyme activities. Whereas shortening the duration of treatment to 5 days during the peripubertal and adult periods resulted in only a partial masculinization of these activities, reducing the dosage of testosterone enanthate from 5 mumol/kg/day to 2.5 mumol/kg/day during both the peripubertal (15 days) and adult periods (9 days) still fully masculinized testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities. Northern blot analysis showed that peripubertal and adult testosterone treatment of female rats increased hepatic CYP2C11 mRNA levels, decreased steroid 5 alpha-reductase mRNA levels and did not change CYP2C6 mRNA levels. Enhanced cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxylation was found in liver microsomes isolated from adult female rats exposed to testosterone during puberty and adult life. In contrast to once daily subcutaneous injections, continuous testosterone release via subcutaneous implant was ineffective in producing the long-term changes in testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities. Overall, the present study establishes that peripubertal androgen imprinting of CYP2C11 and steroid 5 alpha-reductase can be achieved after daily subcutaneous testosterone administration. This occurs by a pretranslational mechanism(s), which lead to long-lasting effects on microsomal drug activation.

Cotransmission of classic transmitters at the synapse has been mentioned for both the CNS and the PNS. Neuropeptide Y (NPY) is a cotransmitter in noradrenergic neurotransmission. In an attempt to understand the heteroregulation of norepinephrine (NE) and NPY biosynthesis, the present study was performed using radioimmunoassay of NPY and northern blotting of cDNA probes for characterization of NPY mRNA. Values of NPY-like immunoreactivity (NPY-ir) were elevated in the cerebrocortex from rats that received treatment with fusaric acid, an inhibitor of dopamine-beta-hydroxylase, with a parallel decrease in NE. Similar results were also observed in rats treated with DSP-4, an alkylator of vesicles in noradrenergic nerve terminals. Moreover, cerebrocortical NPY-ir was reduced in rats receiving treatment with pargyline, an inhibitor of monoamine oxidase, with an elevation of catecholamine in parallel. Activity of NPY mRNA was modified by these drugs in a similar way. However, values of NPY-ir and NE in the cerebrocortex were not influenced by treatment with sodium nitroprusside or guanethidine at a dose producing hypotensive effect. Mediation of hypotensive reflex can thus be ruled out. The data obtained suggest that in vivo decrease of NE by drugs increases biosynthesis of NPY in the cerebrocortex of rats.

Expression of sex-dependent rat hepatic cytochromes P450 and steroid 5 alpha-reductase is regulated mainly by the sex-specific pattern of growth hormone (GH) secretion and is subject to androgen imprinting. Since tamoxifen suppresses GH pulse amplitude and nadir levels, we investigated the effect of tamoxifen on peripubertal testosterone imprinting of hepatic CYP2C11, CYP3A2, CYP2A1, and steroid 5 alpha-reductase. Prepubertal tamoxifen administration (5 mg once daily s.c. on days 28 and 29 of age) to non-ovariectomized female Sprague-Dawley rats did not affect hepatic microsomal CYP2C11-dependent testosterone 2 alpha-hydroxylase, CYP3A-mediated testosterone 6 beta-hydroxylase, CYP2A1-dependent testosterone 7 alpha-hydroxylase, or steroid 5 alpha-reductase activity in adult rats. Testosterone treatment (5 mumol/kg, s.c., once daily) of intact female rats during either puberty (days 35-49 of age) or adult life (days 69-77 of age) had no effect on these enzyme activities in adult (78-day-old) female rats, but the same treatment given during both of these periods induced the male-specific testosterone 2 alpha- and 6 beta-hydroxylase activities and suppressed the female-predominant testosterone 7 alpha-hydroxylase and steroid 5 alpha-reductase activities, indicating that peripubertal testosterone administration imprints the adult androgen responsiveness but not the basal levels of these enzyme activities in non-ovariectomized female rats. However, peripubertal androgen imprinting of the basal levels of testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities was observed in female rats administered tamoxifen prepubertally. Tamoxifen pretreatment also enhanced testosterone imprinting of the adult androgen responsiveness of testosterone 2 alpha- and 6 beta-hydroxylase and steroid 5 alpha-reductase activities. The enhanced testosterone hydroxylase activities were, however, not associated with an increase in microsomal NADPH-cytochrome P450 reductase activity, but were accompanied by elevated hepatic CYP2C11 and CYP3A2 protein levels. Overall, the present study indicates that prepubertal tamoxifen administration does not interfere with the normal sex differentiation of the gender-dependent hepatic cytochromes P450 and steroid 5 alpha-reductase, but this drug modulates peripubertal androgen imprinting of CYP2C11, CYP3A2, and steroid 5 alpha-reductase in adult female rats.

Intracranial germinoma associated with neurofibromatosis 1 (NF-1) has never been documented previously. We report a case of familial NF-1 with a germinoma involving the right basal ganglion and thalamus. A 12-year-old boy presented with multiple café-au-lait spots and a family history of neurofibromatosis in his mother, one of two siblings, and his maternal grandfather. His intracranial lesion was subtotally resected. Histologically, it was a pure germinoma. Serum alpha-feto protein and beta-human chorionic gonadotropin levels were within the normal range. Postoperative myelographic examination and cerebrospinal fluid cytology study showed no evidence of subarachnoid seeding. The patient received postoperative combination chemotherapy resulting in complete response and clearance of the residual tumor. Although this finding of an intracranial germinoma in a patient with familial NF-1 may be coincident, it is suggestive of a potential genetic predisposition. Longitudinal evaluation for the possibility of neoplasm, especially germ cell tumor, in basal ganglion lesions in NF-1 patients is necessary.

The membrane-bound endogenous fatty acid arachidonic acid can be released from membranes by phospholipases and then metabolized to biologically active compounds by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) enzymes. In the liver the CYP pathway is the most significant. Liver CYP arachidonate products include epoxyeicosatrienoic acids (EETs) and monohydroxylated products (HETEs). We examined metabolism of [1-14C]arachidonic acid by a panel of 10 human CYP enzymes expressed in HepG2 cells. In the absence of expressed CYP enzymes, control HepG2 cell microsomes generated only small amounts of omega- and omega--1-OH arachidonic acid (ratio 2:1). Microsomes from HepG2 cells expressing CYP2C8, 2C9, 1A2, and 2E1 were 7-21 times more active than microsomes from the HepG2 controls. CYP2C8, 2C9, and 1A2 principally generated epoxygenase products; 36 to 48% were in the form of EET-diols, reflecting host HepG2 microsomal epoxide hydrolase activity. CYP2C8 and 2C9 formed more 14,15- and 11,12-EET than did CYP1A2, while CYP1A2 formed more 8,9-EET. CYP2C9 also generated a peak with the retention time of 12-HETE. CYP2E1 generated omega--1-OH arachidonic acid and, to a lesser extent, omega-OH arachidonic acid (ratio 2:1). A small amount of epoxygenase activity was also detected for CYP2B6; its overall activity, however, was only about twice control levels. Activities of CYP2A6, 3A3, 3A4, and 3A5 were low and limited to the omega-/omega--1-OH arachidonic acid peak; CYP2D6 was inactive. Microsomes prepared from three individual human livers varied threefold in total arachidonic acid metabolism. For all three livers omega-OH arachidonic acid was the major product (up to 74% of total metabolites). Epoxygenase products constituted 14 to 28% of the total products; 60 to 83% of those were EET-diols, indicating that the human liver microsomes have substantial EET-epoxide hydrolase activity. 11,12-EET was the major EET for two livers and 14,15-EET for the third. The CYP2C inhibitor sulfaphenazole depressed human liver microsomal epoxygenase activity by 50% at 50 microM, while alpha-naphthoflavone inhibited arachidonic acid epoxygenase activity by 27% at 2 microM and by 32% at 10 microM. Collectively, these findings suggest that human liver microsomal arachidonic acid metabolism is catalyzed principally by CYP2C enzymes. CYP1A2, CYP2E1, and possibly CYP2B6 are likely to play more minor roles, though their contribution may be enhanced by exposure to inducers of those enzymes. CYP2A6, CYP2D6, and CYP3A enzymes are unlikely to make any significant contribution.(ABSTRACT TRUNCATED AT 400 WORDS)

The anticancer drug and alkylating agent thiotepa is metabolized by oxidative desulfuration to yield the alkylating metabolite N,N',N"-triethylenephosphoramide (TEPA) in a reaction that is catalyzed by specific liver cytochrome P450 (CYP) enzymes, including CYP2B1, the major phenobarbital-inducible P450 of rat liver, and CYP2C11, a constitutively expressed, male-specific form. The present study investigates the potential for modulating the cytotoxicity and antitumor activity of thiotepa by prior treatment of tumor-bearing rats with the CYP2B1 inducer phenobarbital or the CYP2C11 inhibitor 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) and examines the role of TEPA in the cytotoxicity of thiotepa in vivo. Administration of thiotepa to adult male rats bearing 9L gliosarcoma, grown s.c., resulted in dose-dependent cytotoxicity (ED90 approximately 12 mg/kg i.v., single dose), as determined by a tumor excision/in vitro colony formation assay carried out 24 hr after drug treatment. Tumor growth delay experiments revealed that thiotepa (5 mg/kg) inhibited 9L tumor growth over a 5- to 7-day period after alkylating agent treatment and this effect was accompanied by moderate body weight loss. Pretreatment with phenobarbital, under conditions in which liver CYP2B1 levels and liver microsomal thiotepa desulfuration to yield TEPA are both markedly increased, did not alter thiotepa's short-term (24-hr) cytotoxicity, as judged by a tumor excision assay, nor did it affect the extent of bone marrow toxicity associated with drug treatment. However, phenobarbital did block the tumor growth delay effect of thiotepa and it also attenuated the body weight loss that occurred during the first 5 days after drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

We have treated 13 children with primary intracranial germ cell tumors with the combination of vinblastine, bleomycin, cisplatin, and etoposide, the so-called VBPE regimen in the past seven years at one institution. The clinical diagnosis of these children were: three germinomas, seven non-germinomatous germ cell tumors, and three recurrent non-germinomatous germ cell tumors without previous chemotherapy. Of this group, three germinomas and three non-germinomatous germ cell tumors were treated primarily with VBPE plus radiotherapy. All of them responded completely, and were free of tumor. Primary VBPE regimen without radiotherapy was used in another four non-germinomatous germ cell tumors. After six courses of VBPE regimen, only one of the four patients achieved complete response for 30 months. VBPE regimen was used as salvage chemotherapy in three recurrent non-germinomatous germ cell tumors without previous chemotherapy. After six courses of VBPE regimen, two had complete response for 58+ months and 78+ months respectively. We concluded that in newly-diagnosed intracranial non-germinomatous germ cell tumors, the combination of chemotherapy VBPE regimen and radiotherapy led to good results. Primary chemotherapy with VBPE alone was not adequate for the treatment of non-germinomatous germ cell tumors. However, VBPE regimen might be one of the salvage therapies for recurrent germ cell tumors without chemotherapy before relapse.

A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides site-specifically onto the N termini of proteins or peptides in aqueous solution and in high yield. We have produced biotinylated or heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH) by ligating it onto synthetic peptides containing biotin or mercury. Polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides containing sites for PEG modification. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of filamentous phage containing Met-hGH with random N-terminal sequences (substrate phage). To facilitate ligations involving proteins with highly structured or buried N termini, a more stable subtiligase was designed that effectively ligates peptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of subtiligase should expand the possibilities for protein semisynthesis and rational protein design.

1. Skimmianine, kokusaginine and confusameline, three furoquinolines extracted from the leaves of Evodia merrillii (Rutaceae), were investigated to characterize their selective effects on subtypes of 5-hydroxytryptamine (5-HT) receptors. 2. In the isolated membranes of rat cerebrocortex, using [3H]-5-HT and [3H]-ketanserin as radioligands, skimmianine and the two other furoquinolines displaced radioligand bindings in a concentration-dependent manner. Lower concentrations were required to affect [3H]-ketanserin binding than [3H]-5-HT binding in the order skimmianine > kokusaginine > confusameline. 3. Furoquinolines inhibited 5-HT-induced contraction mediated by 5-HT2 receptors in the presence of methiothepin in rat isolated aorta. Also, the combination of furoquinolines with ketanserin showed an additive antagonism. 4. These furoquinolines were inactive on the 5-carboxamidotryptamine-induced relaxation of guinea-pig ileum, a 5-HT1-mediated event. However, 5-HT-induced contraction via 5-HT2 receptors was reduced by these furoquinolines in a way similar to that in blood vessels. 5. The failure of these compounds to affect the 5-HT-induced Bezold-Jarisch-like reflex in anaesthetized rats, the major 5-HT3-mediated action, ruled out an action on 5-HT3 receptors. 6. The results obtained suggest that three furoquinoline alkaloids may act on 5-HT receptors in animals, more selectively to the 5-HT2 subtype, in the order of skimmianine > kokusaginine > confusameline.

The alkylating anticancer drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; lomustine) is frequently administered to cancer patients as part of a combination chemotherapy regimen. Previous studies have indicated that CCNU treatment of adult male rats leads to prolonged decreases in liver cytochrome P450 (CYP)-mediated enzyme activities. Because the alkylating agent prodrugs cyclophosphamide and ifosphamide are known to be activated by liver cytochrome P450 enzymes, the potential for interaction between CCNU and these oxazaphosphorines was examined. Treatment of adult male rats with a single dose of CCNU (30 mg/kg i.p.) resulted in a progressive loss of liver microsomal cyclophosphamide and ifosphamide hydroxylation activities in vitro (30-60% decrease after 7-27 days). The individual liver P450 forms modulated by CCNU were then identified using P450 form-specific microsomal testosterone hydroxylase assays. CCNU treatment was found to decrease substantially CYP2C11-dependent testosterone 2 alpha-hydroxylase activity (80-90% decrease after 14 or 27 days), but it did not affect CYP3A2-dependent testosterone 6 beta-hydroxylase activity. It only modestly decreased CYP2A2-mediated testosterone 15 alpha-hydroxylase activity. The reduction in CYP2C11 activity was not associated with a decline in liver microsomal NADPH-cytochrome P450 reductase activity, but rather was caused by a complete suppression of CYP2C11 mRNA levels. In contrast to other alkylating agents, such as cisplatin, which is known to feminize the overall expression profile of gender-specific liver enzymes, CCNU did not increase levels of the female-predominant liver enzymes steroid 5 alpha-reductase and CYP2C7, nor did it deplete circulating testosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A variety of chemicals, including triacetyloleandomycin (TAO), alpha-naphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 microM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC50 = 0.4-0.5 microM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 microM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC50 approximately 125 microM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 microM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other P450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.

The present study identifies the specific human cytochrome P-450 (CYP) enzymes involved in hydroxylation leading to activation of the anticancer drug cyclophosphamide and its isomeric analogue, ifosphamide. Substantial interindividual variation (4-9-fold) was observed in the hydroxylation of these oxazaphosphorines by a panel of 12 human liver microsomes, and a significant correlation was obtained between these 2 activities (r = 0.85, P < 0.001). Enzyme kinetic analyses revealed that human liver microsomal cyclophosphamide 4-hydroxylation and ifosphamide 4-hydroxylation are best described by a 2-component Michaelis-Menten model composed of both low Km and high Km P-450 4-hydroxylases. To ascertain whether one or more human P-450 enzymes are catalytically competent in activating these oxazaphosphorines, microsomal fractions prepared from a panel of human B-lymphoblastoid cell lines stably transformed with individual P-450 complementary DNAs were assayed in vitro for oxazaphosphorine activation. Expressed CYP2A6, -2B6, -2C8, -2C9, and -3A4 were catalytically competent in hydroxylating cyclophosphamide and ifosphamide. Whereas CYP2C8 and CYP2C9 have the characteristics of low Km oxazaphosphorine 4-hydroxylases, CYP2A6, -2B6, and -3A4 are high Km forms. In contrast, CYP1A1, -1A2, -2D6, and -2E1 did not produce detectable activities. Furthermore, growth of cultured CYP2A6- and CYP2B6-expressing B-lymphoblastoid cells, but not of CYP-negative control cells, was inhibited by cyclophosphamide and ifosphamide as a consequence of prodrug activation to cytotoxic metabolites. Experiments with P-450 form-selective chemical inhibitors and inhibitory anti-P-450 antibodies were then performed to determine the contributions of individual P-450s to the activation of these drugs in human liver microsomes. Orphenadrine (a CYP2B6 inhibitor) and anti-CYP2B IgG inhibited microsomal cyclophosphamide hydroxylation to a greater extent than ifosphamide hydroxylation, consistent with the 8-fold higher activity of complementary DNA-expressed CYP2B6 with cyclophosphamide. In contrast, troleandomycin, a selective inhibitor of CYP3A3 and -3A4, and anti-CYP3A IgG substantially inhibited microsomal ifosphamide hydroxylation but had little or no effect on microsomal cyclophosphamide hydroxylation. By contrast, the CYP2D6-selective inhibitor quinidine did not affect either microsomal activity, while anti-CYP2A antibodies had only a modest inhibitory effect. Overall, the present study establishes that liver microsomal CYP2B and CYP3A preferentially catalyze cyclophosphamide and ifosphamide 4-hydroxylation, respectively, suggesting that liver P-450-inducing agents targeted at these enzymes might be used in cancer patients to enhance drug activation and therapeutic efficacy.

Factor VII deficiency is an autosomal recessive hereditary disorder characterized by a normal partial thromboplastin time and a prolonged prothrombin time. Definite diagnosis of this condition requires a specific Factor VII assay. Its quite uncommon occurrence in the newborn was first described by Alexander and associates in 1951. A one-day-old male, full-term newborn was presented here with skin pallor, respiratory distress and bitemporal bulging masses noted immediately after birth. Computed tomography scan of the brain revealed a subgaleal hematoma and subarachnoid hemorrhage. Laboratory studies revealed prolonged prothrombin time and a Factor VII level less than 1%. Literature on the clinical manifestation and management of Factor VII deficiency is reviewed briefly.

Cyclophosphamide treatment of adult male rats leads to sustained decreases in several liver microsomal cytochrome P450 (CYP) activities, including CYP 2C11-catalyzed cyclophosphamide activation, via a process that is associated with a feminization of the overall pattern of liver enzyme expression (G. A. LeBlanc and D. J. Waxman, Cancer Res., 50:5720-5726, 1990). The present study compares the effects of cyclophosphamide and its isomeric analogue ifosphamide on the gender-dependent expression of hepatic CYP 2C11 and steroid 5 alpha-reductase in adult male rats and also examines the role of the cyclophosphamide metabolites acrolein and phosphoramide mustard in feminizing the expression of these liver enzymes. Ifosphamide (a) suppressed the male-specific CYP 2C11 mRNA and CYP 2C11-catalyzed liver microsomal testosterone 2 alpha-hydroxylation and cyclophosphamide and ifosphamide 4-hydroxylation and (b) elevated the female-dominant liver enzyme steroid 5 alpha-reductase and its mRNA 7-9 days after drug treatment, both occurring in a manner similar to that of cyclophosphamide, but requiring a 50% higher dose (180 mg/kg, single i.p. injection) to achieve these effects. This pattern of response could not be achieved by treatment of rats with acrolein or with cyclophosphamide analogues that decompose to acrolein without formation of phosphoramide mustard. In contrast, phosphoramide mustard treatment (100 mg/kg) did modulate microsomal CYP 2C11 and steroid 5 alpha-reductase activities. Treatment with a lower dose (50 mg/kg) of phosphoramide mustard or with the acrolein precursor 4-hydroperoxydechlorocyclophosphamide (200 mg/kg) alone did not affect liver enzyme expression, whereas the combination of these agents produced an overall pattern of response that was similar to that conferred by cyclophosphamide. These studies establish that ifosphamide is less potent than cyclophosphamide in modulating the pattern of cytochrome P450 and steroid 5 alpha-reductase expression and that phosphoramide mustard is responsible for the modulation of liver enzyme expression by cyclophosphamide, with acrolein potentiating the modulating activity of the mustard.

Standard management of malignant brain tumors includes either surgical resection alone or surgery followed by irradiation. However, neuroaxis irradiation administered to very young children for primary intracranial tumors is often associated with major late side effects. To delay irradiation and evaluate the efficacy of preirradiation chemotherapy, we treated 9 young children (aged less than 3 years), who had newly diagnosed brain tumors and underwent total or subtotal resection, with a combination of chemotherapy including vinblastine, cisplatin, and etoposide every 3-4 weeks for 6-14 courses between 1988 and 1992. There were malignant gliomas in four patients, medulloblastomas in three, and ependymomas in two. A response to preirradiation chemotherapy (complete remission or partial remission) occurred in seven out of nine cases. Only one patient had progressive disease during the chemotherapy period. Preirradiation chemotherapy with vinblastine, cisplatin, and etoposide might be a highly effective combination allowing delay of radiation therapy in very young children with brain tumors. Acute and subacute toxicity of chemotherapy in this study was mild.

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.

A 1 year and 7 months old girl had suffered from chronic diarrhea for 6 weeks. A huge abdominal mass was found. She received subtotal resection of the tumor and the histological picture showed ganglioneuroblastoma. Then, 12 courses of chemotherapy (including cyclophosphamide, doxorubicin cisplatin and etoposide) were given for 8 months, but the residual tumor mass over the previous surgical area was still noted by CT scan. A second excision of the tumor was performed and histology showed a picture of ganglioneuroma incidentally. Chemotherapy was discontinued. The patient is still alive and well for more than one year.

Congenital brain tumor is a rare disease in the neonatal period. According to the literatures, they comprise only about 1% of childhood brain tumors. Among the congenital brain tumors, 10%-25% are astrocytomas. Anaplastic astrocytoma is one of the malignant glioma. The prognosis is usually not good in the childhood or adult stage. We report one case of congenital anaplastic astrocytoma who received combination chemotherapy, including vinblastine, cisplatin and etoposide following subtotal resection of tumor. After chemotherapy, he got a favorable outcome. And now, he is still no evidence of tumor recurrence for two years.

Primary astrocyte cultures from neonatal rat brains show a Na+-dependent, desipramine-sensitive uptake of [3H]norepinephrine ([3H]NE). Xylamine, a nitrogen mustard, attenuated this uptake of [3H]NE into the astrocytes (IC50 = 0.8 microM). The dose-dependent reduction of [3H]NE uptake by xylamine indicated competitive kinetics. However, xylamine lost the effect if the active transport inhibitor, ouabain or iodoacetate, was also incubated during the pretreatment period of if Na+ was absent. The activity of Na(+)-K(+)-ATPase in astrocytes was not modified by xylamine at the concentrations sufficient to block the uptake of [3H]NE. These findings suggest that xylamine has the ability to compete with the transport of [3H]NE into astrocytes through an effect on the carrier instead of an action on enzymatic activity.

The effect of toluene on the hypothalamic hormone-secreting neurons and neurotransmitter-containing fibers in the rat was investigated by immunohistochemical methods. Multiple intraperitoneal injections of toluene (totally 7.5 ml) led to significant decreases of the neuronal numbers of vasopressin, oxytocin and neuropeptide Y in the preoptic and hypothalamic areas. The densities of vasopressin, oxytocin, norepinephrine and neuropeptide Y immunoreactive fibers of the toluene dose group decreased markedly in the median eminence. In contrast, LHRH neurons remained unchanged.

Superior vena cava syndrome (SVCS) is rare in children. In Veterans General Hospital-Taipei, a total of 364 cases of SVC syndrome were diagnosed in the past 12 years. Of them only seven cases were younger than 18 years of age, ranging from 6 to 17 years, and they were all caused by mediastinal tumor. The underlying malignancy included malignant lymphoma in 4, teratocarcinoma in one and unknown in 2. The most common initial symptom was cough, followed in order of frequency by chest discomfort or neck mass. Dyspnea, orthopnea, swelling of head and neck, and venous engorgement might develop gradually within one to three weeks. Of the reported seven cases, two cases received immediate resuscitation upon arrival but expired in 1-2 hours. The other five cases received treatment with intravenous steroid as well as chemotherapy, and three cases also received committent emergent radiotherapy. Two of them expired 4 months and 2 years after treatment, respectively. Of the two surviving cases, one has received a complete course of chemotherapy and the other is still under regular chemotherapy in our hospital. Both of them are stable till now.

The influence of peripubertal exposure to physiological doses of testosterone on the adult androgen responsiveness of hepatic microsomal cytochrome P-450 was investigated. Male and female Sprague-Dawley rats were sham-operated or gonadectomized before puberty, at 25 days of age. They were injected subcutaneously with testosterone enanthate (5 mumol/kg/day) during the pubertal time period, on days 35-49. Responsiveness to this same dose of testosterone was tested by administering the compound during adulthood, on days 81-89. The females provided a model that had not been exposed to neonatal androgen imprinting, in contrast to the males. Testosterone 2 alpha-hydroxylase activity and cytochrome P-450IIC11, which are normally expressed only in adult males, were expressed in the gonadectomized females administered testosterone during puberty with no further exposure to the hormone for the next 40 days. The levels found were similar to those in the gonadectomized male group. When the combined pubertal and adult testosterone regimen was used, a synergistic effect was produced; the 2 alpha-hydroxylase activity reached control male levels in both gonadectomized and sham-operated females and, in addition, cytochrome P-450IIC11 attained control male levels in the gonadectomized females. Testosterone 6 beta-hydroxylase and erythromycin N-demethylase activities were used as indicators of the cytochrome P-450IIIA subfamily. These activities were significantly increased only in the females treated with testosterone during both the pubertal and adult periods, reaching control male levels of 6 beta-hydroxylation. A similar effect, but in the opposite direction, was found with testosterone 7 alpha-hydroxylase, an enzyme activity indicative of cytochrome P-450IIA1. A decrease in this enzyme was produced in the females administered testosterone during both time periods, resulting in levels equivalent to those found in control males. In general, a highly significant interaction was found between the pubertal and adult treatment periods for the females, indicating a chronic effect of the pubertal exposure. The experiments with castrated males did not result in synergistic interactions, although there was some evidence of an additive effect. The results of this study support the hypothesis that the peripubertal period is a time during which testosterone imprinting of both increased basal levels and adult androgen responsiveness of some hepatic cytochrome P-450 enzymes can occur in the female rat.

Genes for the blue copper proteins Populus nigra var. italica plastocyanin and Pseudomonas aeruginosa azurin have been constructed by a stepwise procedure. The leader sequence for azurin has been placed before the genes directing plastocyanin and azurin transport to the periplasmic space when the genes are expressed in Escherichia coli. Site-saturation mutagenesis has been used to alter two copper-binding residues of azurin (Met-121 and His-46) and Met-92 of plastocyanin. While the plastocyanin mutants do not appear to bind copper, the azurin variants all bind copper and show characteristic type I blue copper centers. In particular, the electronic spectra reflect the dominance of the charge transfer interaction between copper and the thiolate of Cys-112, being relatively insensitive to changes in Met-121 or His-46. In contrast, removal of Met-121 appreciably alters the EPR spectra of the mutants, although, to a first order, the spectra of all mutants are themselves similar, suggesting a more distorted geometry around copper in the mutants than in the wild type.

A rare case of solitary eosinophilic granuloma of the sternum in a 5-year-old boy is reported. He presented anterior chest pain and a nodular, somewhat tender mass over the sternum. Chest x-ray, tomography, CT scanning of the sternum and total body Gallium scanning revealed an isolated lytic lesion 5 cm below the sternal angle. An excisional biopsy of the mass showed the typical histologic appearance of eosinophilic granuloma. An isolated eosinophilic granuloma of sternum is rarely reported in the English literature. The differential diagnosis of sternal tumors and a review of eosinophilic granuloma of sternum is discussed.

Neutralizing antibody titers to poliovirus type 1, 2 and 3 were examined in paired serum specimens obtained from 43 female hospital personnel and laboratory workers at Veterans General Hospital. Before vaccination, 23.2% of those studied did not have neutralizing antibodies to one or more types of poliovirus, and 4.6%, 13.9% and 7.0% lacked serum neutralizing antibodies to poliovirus type 1, 2 and 3, respectively. In a comparison of the serological responses evoked by oral poliovirus vaccine in the same persons, a striking difference in antibodies was shown in the geometric mean titers and no lack of antibodies was found after oral poliovaccine immunization. Therefore immunization with oral poliovirus vaccine should be considered in the future as to improved protection for such high risk people.

Fourteen commercially available monoclonal antibodies were used to examine, by the indirect immunoperoxidase technique, 20 formaldehyde solution-fixed, routinely processed bone marrow biopsy and surgical biopsy specimens from a group of small round cell tumors with bone marrow involvement, including 6 neuroblastomas, 4 rhabdomyosarcomas, 3 Ewing's sarcomas, 3 Burkitt's lymphomas, and 4 small-cell carcinomas of the lung. Eight of 14 monoclonal antibodies worked well on the routinely processed sections. Antidesmin (D33) (Dako Corp, Santa Barbara, Calif) and anti-muscle actin (HHF35) showed specific immunostaining in all 4 rhabdomyosarcomas included in this study. Anti-epithelial membrane antigen immunoreactivity was positive in all small-cell carcinomas of the lung. Anti-leukocyte common antigen (Dako) showed positive immunostaining in lymphomas and did not immunoreact with nonhematopoietic tumors. However, it was important to be aware that some lymphomas may not have been labeled with anti-leukocyte common antigen. All 6 neuroblastomas showed a positive immunoreactivity with anti-chromogranin A (LK2H10) and a weak reaction with antisynaptophysin (SY38). Panels of commercially available monoclonal antibodies, including anti-leukocyte common antigen, anti-epithelial membrane antigen, antidesmin (Dako), anti-muscle actin, and anti-chromogranin A, appear to be most useful in the differential diagnosis and staging of small round cell tumors on routinely processed biopsy specimens.

From January 1981 to January 1986, 34 children between 2 and 14 years of age were diagnosed as non-Hodgkin's lymphoma by the Rapport's classification at the Department of Pediatrics of Veterans General Hospital, Taipei, R.O.C. These patients were retrospectively analysed. There was male predominance with a male to female ratio of 2.4:1. The most common symptom was the palpable mass (29 cases, 85.3%) involved the neck and abdomen equally. All the cases were diffuse patterns, DUL (diffuse undifferentiated lymphoma) and DPDL (diffuse poorly differentiated lymphoma) both were the common subtypes of histological patterns. Twenty-eight cases (82%) were in the advanced stage (stage III and IV). The incidence of BM and CNS involvement was 56% and 29.4%, respectively. CNS involvement demonstrated a higher mortality of 100%. Surgery, radiation, and chemotherapy were mainly used for stage I-II and chemotherapy for stage III-IV. The 3-year survival rate of the whole series was 26.5%, and stage I, II, III, and IV was 100%, 50%, 33%, 9%, respectively. The causes of death were CNS involvement (10 cases, 40%), extensive involvement (7 cases, 28.0%), and sepsis (3 cases, 12.0%). This study shows that NHL in children are usually diffuse rather than follicular in histological patterns, often present a palpable mass involved the cervical or abdominal area, and commonly are widespread and tend to disseminate early.