The objective of this study was to synthesize and characterize new energetic polycyclic "cage" compounds. As part of a program involved in the synthesis of new polynitropolycyclic compounds, 2,6-dinitro-5-methoxy- 7-carbomethoxypentacyclo[5. 3 .0 . 0* • * . CP • i ° . 0* •8]decane has been synthesized. This is a model system which can be used to study (1) the effect of nitro substitution on the photolability of carbon-carbon double bonds and (2) to develop methods for avoiding Haller-Bauer cleavage in cage /3-keto esters when synthesizing polynitro-substituted cage compounds.

Hydramycin is an antitumor antibiotic isolated from Streptomyces violaceus. It is a pyranoanthraquinone-type antitumor agent that has shown broad-spectrum activity against a variety of human-derived cancer cell lines. Among tumors evaluated at the National Cancer Institute (lung, colon, melanoma, breast and prostate), GI50s were <10−10 M in the NCI's 60-cell-line panel. We embarked on the synthesis and evaluation of a simplified congener 2-(1-hydroxy-1-(oxiran-2-yl)ethyl)-4H-naphtho[2,3-h]chromene-4,7,12-trione(17), which would facilitate synthesis while retaining the potent activity. Hydramycin has two chiral centers, and our goal is to design and synthesize all the possible enantiomers (four in total) for the congener of hydramycin 17 in order to ascertain which of the enantiomers is responsible for the observed antitumor activity. The use of enantiospecific techniques such as the Sharpless epoxidation was initially tried to introduce the chiral centers at a later stage during the multi-step synthesis and obtain the required pure enantiomers. Due to some limitations observed with this technique and many other asymmetric epoxidation techniques which utilize very substrate-specific ligands, we then modified the synthetic scheme to use another procedure, the Sharpless asymmetric dihydroxylation in order to obtain two of the pure enantiomers of this congener of hydramycin. The other two enantiomers are selectively obtained using the Sharpless asymmetric dihydroxylation procedure via cyclic sulfate intermediates, followed by a modified irreversible Payne rearrangement procedure. These routes then allowed us to obtain separately all four stereoisomers, each having more than 90% ee as determined on chiral columns with HPLC. The four stereoisomers have been fully characterized and will then be tested separately to ascertain which of the isomers is responsible for the observed antitumor activity.

Made available in DSpace on 2014-06-11T19:35:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2000-04-27Bitstream added on 2014-06-13T19:05:07Z : No. of bitstreams: 1 poppi_nr_dr_araiq.pdf: 2732054 bytes, checksum: 0800f7d3640b4b73c285c261aa5734ff (MD5) / município de Campo Grande, MS, com uma população de aproximadamente 600 mil habitantes concentrados na área urbana, todos os anos, entre os meses de julho a novembro, período muito seco, fica encoberto pela fumaça proveniente da queima de biomassa, a qual tem sido apontada como responsável pelo aumento da incidência de casos de doenças respiratórias na população. Neste trabalho, é reportada a composição química de aerossóis, partículas inaláveis (1 μm ≤ dp≤ 15 μm), de emissões do forno utilizado para a produção de carvão vegetal e de 14 amostras de ar da cidade de Campo Grande / MS. O particulado atmosférico foi amostrado de junho a novembro de 1998 no campus da UFMS e as amostras de emissão direta foram coletadas a 1,5 m de um forno construído com tijolos e saibro, similar aos utilizados nas carvoarias da região. Nos dois tipos de amostra, o material particulado foi coletado sobre filtro de Fluorepore em PTFE com 37 mm de diâmetro e as substâncias semivoláteis em tubos do adsorvente XAD-2, utilizando-se amostrador de baixo volume (low-vol). As substâncias foram extraídas com diclorometano/metanol (4:1) em banho ultra-sônico. As análises foram realizadas por CG/EM nos modos SCAN e SIM. A concentração de HPAs na atmosfera próxima ao forno, proveniente da queima de madeira para produção de carvão, foi estimada em 23,6 μg.m-3 de ar para a soma de 15 HPAs, e em 310,1 ng.m-3 para o BaP. A concentração média dos 15 HPAs nas amostras ambientais foi de 21,05 ng.m-3 e a concentração média do BaP de 0,25 ng.m-3... / Campo Grande City (Mato Grosso do Sul State, Brazil) has around 600,000 inhabitants, concentrated in the urban area. Every year, between July and November, the dry season, the city is covered with biomass burning smoke. The biomass burning is result of the vegetal carbon production, agricultural handling, or de-florestation operation into Brazilian Savannah (“cerrado”). The smoke has been indicated as main cause of respiratory disease increasing of the population. In this work it was reported the aerosol composition (1μm ≤ dp≤ 10 μm), from emission of vegetal carbon production and 14 air samples of Campo Grande city. The particulate material was sampled from June until November, 1998 at campus of Federal University of Mato Grosso do Sul (UFMS) while the emission samples were collected at 1.5 m far from an oven made with brick and a mixture of clay and sand, similar to ones used by vegetal carbon producers of region. For both kind of samples, the particulate material was collected with Fuorepore/PTFE filters (37 mm diameter) while volatile fraction was sampled into adsorbent tubes (XAD-2), using low vol sampler. The extracts were obtained by ultrasonic bath using dichloromethane:methanol (4:1) and were analyzed by GC/MS, SCAn and SIM modes. The total HPAs emission of vegetal carbon oven was estimated in 23.6 μg.m-3 and 310 ng.m-3 for benzo(a)pyrene. On the other hand, the city air samples shown concentration (average) of 21,05 ng.m-3 for total HPAs, and 0.25 ng.m-3 for benzo(a) pyrene. HPAs, OXI-HPAs, phenols e Metoxy-phenols were identified...(Complete abstract, click electronic access below)

In this diploma Thesis, a method for the preparation of polycarbocyclic compounds containing all-carbon quaternary centers embedded in triquinane skeleton was prepared. The key reaction sequence leading towards the preparation of these carbocycles was Pd-catalyzed tandem cyclization/Suzuki cross-coupling reaction followed by halocarbocyclization. In the first part of synthetic project, a model oxygen-containing polycyclic derivative was prepared. In the second part, 3 novel polycarbocyclic compounds containing all-carbon quaternary centers were successfully prepared. Synthetic procedure for the starting material required for the key reaction sequence was developed. This approach represents another pathway for the synthesis of polycarbocyclic natural products. Keywords: synthesis, polycyclic compounds, quaternary centers, catalysis

The synthesis of a novel, cage spiro-oxetane was carried out. Pentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecane-8- one (PCUD-8-one) undergoes one-carbon homologation to a mixture of endo- and exo- PCUD-carboxaldehydes which then are converted into 8,8-bis(hydroxymethyl)PCUD. The monotosylate obtained via reaction of 8,8- bis(hydroxymethyl)PCUD with tosyl chloride(1 equivalent) reacts with sodium hydride to afford the corresponding spiro-oxetane via intramolecular Williamson reaction. Six new substituted heptacyclo[6.6.0.0^2,6.0^3,13.0^4,11. 0^5,9.0^10,14]tetradecanes (HCTD) were synthesized. These compounds will be used as substrates in a photoelectron spectroscopic study. The ring-expansion reaction of PCUD-8-one with ethyl diazoacetate in the presence of BF_3:OEt_2 was performed. The major product was converted into an alcohol, and the structure of the 3,5-dinitrobenzoate of this alcohol was elucidated by single crystal x-ray structural analysis.

Several antibiotics that prevent translation by binding to ribosomal subunits have been shown to also inhibit ribosomal subunit assembly (Champney and Tober 2003). The aminoglycoside hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal subunit assembly. The streptogramin antibiotics quinupristin-dalfopristin and XRP 2868 (NXL 103) were examined for similar effects on these 2 cellular functions in antibiotic-resistant strains of Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pulse chase experiments were performed which verified slower rates of ribosomal subunit formation in drug treated cells. Hygromycin B exhibited a concentration dependent inhibitory effect on viable cell number, growth rate, protein synthesis and 30S and 50S subunit formation. 16S rRNA specific probes hybridized to rRNA fragments in cells treated with hygromycin B. RNase II and RNase III deficient strains of E. coli exhibited the most accumulation of 16S rRNA fragments upon treatment with hygromycin B. Examination of total RNA from treated cells showed an increase in RNA corresponding to precursor to the 16S rRNA while 16S rRNA decreased. There was also an increase in small fragment RNA. Hygromycin B was a more effective inhibitor of translation than ribosomal subunit formation in E. coli. Two streptogramin antibiotics were compared for inhibitory effects in antibiotic-resistant Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. IC50 values for XRP 2868 were several fold lower than those of quinupristin-dalfopristin for inhibition of cell viability, protein synthesis, and ribosomal subunit formation. Both antibiotics revealed a concentration dependent inhibitory effect on cellular functions including 50S ribosomal subunit formation in the three organisms examined. XRP 2868 inhibited both 50S ribosomal subunit assembly and translation. XRP 2868 was effective against MRSA and was a better inhibitor in each of the antibiotic resistant strains examined compared with quinupristin-dalfopristin.

En aquesta Tesi: 1) S’ha estudiat la reacció de metàtesi creuada (CM) de diferents derivats metilenciclopentànics utilitzant el catalitzador d’Hoveyda-Grubbs de segona generació, observant la formació dels alquens tetrasubstituïts corresponents amb bons rendiments. En el cas d’un derivat bis(metilenciclopentànic) s’ha observat la formació de productes de doble i triple CM amb elevada estereoselectivitat anti, fet que en el cas del producte de doble CM s’ha establert per difracció de raigs X. 2) S’ha preparat un nou ciclopentadiè 1,1-disubstituit funcionalitzat, i s’han estudiat les reaccions de Diels-Alder amb diferents dienòfils [anhídrid maleic, cis-1,2-bis(fenilsulfonil)etilè i triflat de fenil(2-iodoetinil)iodoni]. L’adducte amb l’últim dienòfil s’ha transformat en un derivat 2,3-diiodat 7,7-disubstituït del norbornadiè. 3) S’ha estudiat la reacció del triflat de (2-iodoetinil)feniliodoni amb 1,3-difenilisobenzofuran que depenen de les condicions dóna l’adducte Diels-Alder corresponent o un triflat naftalènic format per reducció de l’adducte inicial per part del 1,3.difenilisobenzofuran emprat en excés. 4) S’ha desenvolupat una seqüència sintètica per a l’obtenció de derivats del 2,8-etanonoradamantà molt funcionalitzats, que té com a etapa clau una reacció Diels-Alder intramolecular. Durant la reducció d’una enona a alcohol al•lílic amb NaBH4 / CeCl3•7H2O (mètode de Luche), es va haver de protegir una funció maleimida transformant-la en una barreja diastereomèrica d’adductes amb furà, regenerant la maleimida després de la reducció de l’enona, per escalfament en el sí de toluè a reflux durant 4 dies (reacció retro-Diels-Alder). També s’ha posat a punt un procediment alternatiu per preparar un intermediari clau en la seqüència sintètica anterior, 5-{[(t-butildimetilsilil)oxi]metil}-2-metil-5,6-dihidrociclopenta[c]pirrol-1,3(2H,4H)-diona, a partir de la N-metilmaleimida amb un rendiment cinc vegades superior al desenvolupat inicialment. 5) L’estructura dels diferents compostos obtinguts en aquesta Tesi s’ha establert per mètodes espectroscòpics [1H-RMN, 13C-RMN, correlacions 1H/1H (COSY, NOESY), correlacions 1H/13C (gHSQC, gHMBC), MS, IR] i en diversos casos, també per difracció de raigs X. / "Synthesis and reactivity of polycyclic compounds: Applications of the olefin metathesis reaction to their synthesis" In this PhD Thesis: 1) The cross metathesis (CM) reaction of different methylenecyclopentane derivatives using Hoveyda-Grubbs second generation catalyst has been studied. In these reactions, tetrasubstituted alkenes have been formed in good yields. In the case of a bis(methylenecyclopentane) derivative, the formation of products from double and triple CM has been observed. In general, high anti-stereoselectivity has been observed. The assignment of the stereochemistry of the single CM products has been performed through the analysis of the NMR data for the epoxide derivatives and in the case of the double CM product by X-ray diffraction analysis. 2) A new 1,1-disubstituted cyclopentadiene has been prepared. Its reactions with different dienophiles [maleic anhydride, cis-1,2-bis(phenylsulfonyl)ethylene and phenyl(2-iodoethynyl)iodonium triflate] has been studied. The adduct with the last dienophile has been transformed into a 2,3-diiodo-7,7-disubstituted norbornadiene. 3) The reaction of phenyl(2-iodoethynyl)iodonium triflate with 1,3-diphenylisobenzofuran has been studied. Depending on the reaction conditions, the corresponding Diels-Alder adduct or a naphthalenic triflate, formed by reduction of the initial adduct with 1,3-diphenylisobenzofuran used in excess, has been observed. 4) A synthetic sequence for the preparation of highly functionalized 2,8-ethanonoradamantane derivatives has been developed. The key step consists of an intramolecular Diels-Alder reaction. On reduction of an enone function to an allylic alcohol using NaBH4 / CeCl3•7H2O (Luche procedure), a maleimide function should be protected by transformation into a stereoisomeric mixture of Diels-Alder adducts with furan. The maleimide function was recovered, after reduction of the enone, through a thermal retro-Diels-Alder reaction. Also, an alternative procedure to prepare a key intermediate of the above synthetic sequence, 5-{[(t-butyldimethylsilyl)oxy]methyl}-2-methyl-5,6-dihydrocyclopenta[c]pyrrole-1,3(2H,4H)-dione, from N-methylmaleimide with a yield five times higher, have been developed. 5) The structure of the different compounds prepared in this PhD Thesis has been established by spectroscopic methods [1H NMR, 13C NMR, 1H/1H correlations (COSY, NOESY), 1H/13C correlations (gHSQC, gHMBC), MS, IR] and, in several cases, also by X-ray diffraction analysis.

The large conductance Ca++-activated K+ channel (BKCa) regulates neuronal excitability through the efflux of K+, in response to membrane depolarization and increases in intracellular Ca++. The activity of the BKCa channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie, in part, the influence of the drug on peptide hormone release from neurohypophysial nerve terminals (Dopico et al., 1996, 1998). Moreover, chronic EtOH exposure attenuates acute drug action on hormone release, and reduces the sensitivity of BKCa channels to acute EtOH exposure (Knott et al., 2002). The factors regulating EtOH action on BKCa channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels, including BKCa (Chang et al., 1995a, b; Moczydlowski et al., 1985; Park et al., 2003; Turnheim et al., 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distribution of membrane cholesterol (CHS) (Chin et al., 1978; Chin et al., 1979; Wood et al., 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKCa channel activity and EtOH sensitivity. To address this, we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKCa channels retain EtOH sensitivity in this reductionist preparation (Chu et al., 1998), and we extend the study here to examine cloned human brain (hslo) BKCachannels. We show here that hslo channels maintain their characteristic large conductance, voltage and Ca++-dependent gating, and sensitivity to 50 mM EtOH in bilayers cast from a 3:1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels, in a concentration-dependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings, we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKCa channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structure itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore, reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel, we demonstrate that decreases in negative surface charge, increases in the proportion of unsaturated acyl chains, and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslochannels, relative to control POPE/POPS (3:1) bilayers. Overall, these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S.N., O'Connell, R.J., and Crowley, J.J. (2002). Artificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research, D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J.J., Treistman, S.N., and Dopico, A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKCA channels in binary phospholipid bilayers. Mol. Pharma. 64(2):364-372.

Orientador: Mary Rosa Rodrigues de Marchi Santiago da Silva / Banca: Lilian Rotschild de Carvalho / Banca: Fernando Mauro Lanças / Banca: Maria Lucia Ribeiro / Banca: Alberto José Cavalheiro / Resumo: município de Campo Grande, MS, com uma população de aproximadamente 600 mil habitantes concentrados na área urbana, todos os anos, entre os meses de julho a novembro, período muito seco, fica encoberto pela fumaça proveniente da queima de biomassa, a qual tem sido apontada como responsável pelo aumento da incidência de casos de doenças respiratórias na população. Neste trabalho, é reportada a composição química de aerossóis, partículas inaláveis (1 μm ≤ dp≤ 15 μm), de emissões do forno utilizado para a produção de carvão vegetal e de 14 amostras de ar da cidade de Campo Grande / MS. O particulado atmosférico foi amostrado de junho a novembro de 1998 no campus da UFMS e as amostras de emissão direta foram coletadas a 1,5 m de um forno construído com tijolos e saibro, similar aos utilizados nas carvoarias da região. Nos dois tipos de amostra, o material particulado foi coletado sobre filtro de Fluorepore em PTFE com 37 mm de diâmetro e as substâncias semivoláteis em tubos do adsorvente XAD-2, utilizando-se amostrador de baixo volume (low-vol). As substâncias foram extraídas com diclorometano/metanol (4:1) em banho ultra-sônico. As análises foram realizadas por CG/EM nos modos SCAN e SIM. A concentração de HPAs na atmosfera próxima ao forno, proveniente da queima de madeira para produção de carvão, foi estimada em 23,6 μg.m-3 de ar para a soma de 15 HPAs, e em 310,1 ng.m-3 para o BaP. A concentração média dos 15 HPAs nas amostras ambientais foi de 21,05 ng.m-3 e a concentração média do BaP de 0,25 ng.m-3...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Campo Grande City (Mato Grosso do Sul State, Brazil) has around 600,000 inhabitants, concentrated in the urban area. Every year, between July and November, the dry season, the city is covered with biomass burning smoke. The biomass burning is result of the vegetal carbon production, agricultural handling, or de-florestation operation into Brazilian Savannah ("cerrado"). The smoke has been indicated as main cause of respiratory disease increasing of the population. In this work it was reported the aerosol composition (1μm ≤ dp≤ 10 μm), from emission of vegetal carbon production and 14 air samples of Campo Grande city. The particulate material was sampled from June until November, 1998 at campus of Federal University of Mato Grosso do Sul (UFMS) while the emission samples were collected at 1.5 m far from an oven made with brick and a mixture of clay and sand, similar to ones used by vegetal carbon producers of region. For both kind of samples, the particulate material was collected with Fuorepore/PTFE filters (37 mm diameter) while volatile fraction was sampled into adsorbent tubes (XAD-2), using low vol sampler. The extracts were obtained by ultrasonic bath using dichloromethane:methanol (4:1) and were analyzed by GC/MS, SCAn and SIM modes. The total HPAs emission of vegetal carbon oven was estimated in 23.6 μg.m-3 and 310 ng.m-3 for benzo(a)pyrene. On the other hand, the city air samples shown concentration (average) of 21,05 ng.m-3 for total HPAs, and 0.25 ng.m-3 for benzo(a) pyrene. HPAs, OXI-HPAs, phenols e Metoxy-phenols were identified...(Complete abstract, click electronic access below) / Doutor

Bile acid metabolism is integrally involved in cholesterol homeostasis in mammals because it is the major means by which cholesterol is eliminated from the body. We have undertaken an effort to study the molecular mechanisms underlying the regulation of bile acid metabolism by isolating and characterizing the cDNA and gene for an enzyme that hydroxylates lithocholic acid (LCA) at position 6β, lithocholic acid 6β-hydroxylase; the first bile acid-induced gene reported. LCA is a very hydrophobic, toxic bile acid formed from chenodeoxycholic acid in the gut lumen upon reduction of the 7α-hydroxy group by microbial enzymes. The proper elimination of LCA is essential for maintenance of the bile acid pool and for prevention of cholestasis which results from LCA precipitating in the cannaculi of the liver when its concentration is high. The LCA 6β-hydroxylase cDNA was isolated by differential hybridization of hamster liver libraries prepared from animals fed either a cholic acid enriched diet or a cholestipol-rich chow and was named CYP3A10 based on its homology with other cytochrome P450s (P450) in family 3A. We found that CYP3A10 was essentially expressed only in males. A statistical analysis of RNA from young males fed with cholic acid and normal chow showed that the cholic acid induction was about 50% at the RNA level. We determined the biological nature of the protein encoded by CYP3A10 by expression of the cDNA in COS cells. Microsomes prepared from transfected cells were assayed with LCA as a substrate and found to hydroxylate LCA predominantly at position 6β. We examined whether CYP3A10 could hydroxylate other steroid compounds by assays with testosterone, progesterone and androstenedione and found that, although 6β-hydroxylase (as well as others) activity was observed with all three, LCA was the preferred substrate based on kinetic analysis. A developmental time course of CYP3A10 expression in males showed little expression before puberty, a striking induction of expression at puberty and a fourfold induction thereafter through adulthood. We then examined the male-specific expression of CYP3A10 in hamster liver. We disrupted the pattern of GH secretion in male hamsters by hypophysectomy, neonatal glutamate treatment and by continuous infusion of GH via osmotic minipumps (to mimic the female pattern of GH secretion) and found no significant effect on CYP3A10 expression. Conversely, in females, hypophysectomy and neonatal glutamate treatment significantly induced CYP3A10 expression 5- to 10-fold. Additionally, when females treated neonatally with glutamate were injected twice daily with GH as adults (to mimic the male pattern of GH secretion), the levels of CYP3A10 expression were not significantly different from those of normal males. These results led us to conclude that the pattern of GH secretion in males does not control the male-specific expression of CYP3A10 but that in females expression can be induced by altering the tonic secretion of GH. No significant effect on CYP3A10 expression was observed by castration of adult males, indicating that circulating androgens were not required for expression. We found that gonadal hormones (e.g. estrogen and progesterone) do not have a suppressive effect on CYP3A10 expression in females since ovariectomy did not induce expression. Many genes are "imprinted" neonatally by exposure to a given effector for developmental-, tissue- or sexually regulated expression. We investigated whether neonatal androgen exposure was required for male-specific expression of CYP3A10 by castrating hamsters neonatally and determining the level of CYP3A10 expression in adulthood. Our results indicate that androgens are required neonatally for CYP3A10 expression since no expression was observed in neonatally castrated hamsters. We were unable to induce expression in neonatally castrated hamsters by either GH or testosterone injections. These results suggest several notable points 1) that CYP3A10 expression is programmed neonatally by androgen exposure; 2) that androgens exert their effect directly on the liver and not via the hypothalamus; 3) that neither testosterone nor GH can restore CYP3A10 expression when males have not been exposed to androgens neonatally; and 4) that in experimental conditions, females can be induced to express CYP3A10, which indicates that there are two modes for regulating expression: by "imprinting" in males and by GH and testosterone in females. We are now studying the molecular mechanisms involved in the bile acid-mediated induction and the male-specific expression of CYP3A10. We have cloned approximately 8 kb of 5' flanking DNA from a hamster genomic library and sequenced about 1 kb of proximal DNA. Primer extension and S1 digestion analyses indicate that the mRNA for CYP3A10 has multiple transcription initiation sites clustered about 90 bp from the initiator methionine codon. We have also prepared CYP3A10 promoter/lacZ chimeric constructs to begin delineating the cis-acting elements controlling CYP3A10 expression and regulation. We used H2.35 cells as recipients because they are a mouse hepatocyte cell line that has been transformed with a temperature sensitive SV40. These cells can be grown at the permissive temperature and can be induced to behave like liver cells, the differentiated condition, by switching to a nonpermissive temperature. We have found that the construct with 1 kb of proximal CYP3A10 5' flanking DNA was able to express the reporter gene at higher levels under differentiated conditions, which were consistent with higher expression of an albumin promoter/lacZconstruct, upon switching the cells to the more liver phenotype. The system characterized and described here is ideally suited for dissecting the molecular details governing bile acid-mediated regulation and sexually dimorphic expression of liver genes. Very little is known about both these very important biological phenomena. Much could be learned about transcriptional regulation of liver genes by investigating the cis-elements and trans-acting factors mediating regulation of CYP3A10 expression.