Ultrasensitive PSA: rethinking post-surgical management for node positive prostate cancer.

Introduction: Clinicians may offer patients with positive lymph nodes (pN1) and undetectable PSA following surgery for prostate cancer either observation or adjuvant therapy based on AUA, EAU, and NCCN guidelines considering standard PSA detection thresholds of <0.1ng/ml. Here we sought to investigate the outcomes of pN1 patients in the era of ultrasensitive PSA testing. Methods: We queried the Northwestern Electronic Data Warehouse for patients with prostate cancer who were pN1 at radical prostatectomy and followed with ultrasensitive PSA. Patients receiving neoadjuvant treatment were excluded. We compared clinical characteristics including age, race, pre-operative PSA, Gleason grade, tumor stage, surgical margins, and nodal specimens to identify factors associated with achievement and maintenance of an undetectable PSA (defined as <0.01 ng/mL). Statistics were performed using t-test, Mann-Whitney U test, chi-squared analysis, and logistic regression with significance defined as p<0.05. Results: From 2018-2023, 188 patients were included. Subsequently, 39 (20.7%) had a PSA decline to undetectable levels (<0.01 ng/mL) post-operatively at a median time of 63 days. Seven percent of these men (3/39) were treated with adjuvant RT + ADT with undetectable PSA levels. 13/39 (33.3%) had eventual rises in PSA to ≥0.01 ng/mL for which they underwent salvage RT with ADT. Overall, 23/39 (59%) patients achieved and maintained undetectable PSA levels without subsequent therapy at median follow-up of 24.2 mo. Compared to patients with PSA persistence after surgery or elevations to detectable levels (≥0.01 ng/mL), patients who achieved and maintained undetectable levels had lower Gleason grades (p=0.03), lower tumor stage (p<0.001), fewer positive margins (p=0.02), and fewer involved lymph nodes (p=0.02). On multivariable analysis, only primary tumor (pT) stage was associated with achieving and maintaining an undetectable PSA; pT3b disease was associated with a 6.6-fold increased chance of developing a detectable PSA (p=0.03). Conclusion: Ultrasensitive PSA can aid initiation of early salvage therapy for lymph node positive patients after radical prostatectomy while avoiding overtreatment in a significant subset. 20% of patients achieved an undetectable PSA and over half of this subset remained undetectable after 2 years.

Testosterone recovery after androgen deprivation therapy.

PURPOSE: Finite courses of androgen deprivation therapy (ADT) are often utilized in men undergoing treatment for prostate cancer. Previous evidence suggests that timing of testosterone (T) recovery can be variable after ADT. Recently, an oral gonadotropin releasing-hormone (GnRH) antagonist, relugolix, has demonstrated more rapid T recovery than injectable GnRH agonists such as leuprolide. In this study, we sought to evaluate patient characteristics associated with T recovery in patients undergoing ADT of defined duration. MATERIALS AND METHODS: The Northwestern Enterprise Data Warehouse was queried for men with prostate cancer who completed a course of ADT and subsequently had a testosterone lab performed. Testosterone recovery was evaluated for levels that reached above castrate (T > 50 ng/dl), partial recovery (T > 150 ng/dl), and full recovery (T ≥ 300 ng/dl). RESULTS: 388 men who received finite courses of ADT were identified (348 receiving leuprolide, 36 receiving relugolix, and 4 receiving degarelix). In multivariable Cox regression analysis, men who were prescribed GnRH antagonists (HR = 3.74, CI = 2.53-5.53, P ≤ 0.001) and who were younger (HR for 1 year increase in age = 0.96, CI = 0.95-0.98, P < 0.001) were more likely to achieve partial recovery. In a subgroup analysis, men who received extended ADT courses (>12 months) with a GnRH agonist had lower rates of partial T recovery (HR = 0.58, CI = 0.41-0.81, P = 0.001). CONCLUSION: T recovery after ADT is variable with roughly one sixth of men remaining castrate. GnRH antagonist use and younger age are associated with higher rates of T recovery after ADT. Longer ADT courses were associated with worse T recovery rates.

Laser induced breakdown spectroscopy of pure aluminum with high temporal resolution.

We report on a Laser Induced Breakdown Spectroscopy (LIBS) system with a very high temporal resolution, using femtosecond and picosecond pulse laser excitation of pure aluminum (Al). By using a 140 fs Ti:Sapphire laser in an ultrafast optical Kerr gate (OKG), we demonstrate LIBS sampling with a sub-ps time resolution (0.8 ± 0.08 ps) in a 14 ns window. The width of the gating window in this system was as narrow as 0.8 ps, owing to the inclusion of a carbon disulfide (CS(2)) cell, which has a fast response and a large nonlinear coefficient. Furthermore, when using a 100 ps pulsed Nd:YAG laser and a fast photomultiplier tube (PMT) we demonstrate a LIBS system with a nanosecond time resolution (2.20 ± 0.08 ns) in a microsecond window. With this sort of temporal resolution, a non-continuous decay in the Al signal could be observed. After 50 ns decay of the first peak, the second peak at 230 ns is started to perform. Experimental results with such short temporal windows in LIBS, in both nanosecond and microsecond ranges, are important for fast temporal evolution measurements and observations of early continuum emission in materials.

Simultaneous hydrodynamic cavitation and nanosecond pulse discharge plasma enhanced by oxygen injection.

A novel Hydrodynamic Cavitation-Assisted Oxygen Plasma (HCAOP) process, which employs a venturi tube and oxygen injection, has been developed for enhancing the production and utilization of hydroxyl radicals (·OH) in the degradation of organic pollutants. This study has systematically investigated the fluid characteristics and discharge properties of the gas-liquid two-phase body in the venturi tube. The hydraulic cavitation two-phase body discharge is initiated by the bridging of the cavitation cloud between the electrodes. The discharge mode transitions from diffuse to spark to corona as the oxygen flow rate increases. The spark discharge has the highest current and discharge energy. Excessive oxygen results in the change of the flow from bubbly to annular and a subsequent decrease in discharge energy. The effects of cavitation intensity, oxygen flow rate, and power polarity on discharge characteristics and ·OH production were evaluated using terephthalic acid as a fluorescent probe. It was found that injecting 3 standard liter per minute (SLPM) of oxygen increased the ·OH yield by 6 times with only 1.2 times increase in power, whereas<0.5 SLPM of oxygen did not improve the ·OH yield due to lower breakdown voltage. Negative polarity voltage increased the breakdown voltage and ·OH yield due to asymmetric density and pressure distribution in the throat tube. This polarity effect was explained by numerical simulation. Using indigo carmine (E132) as a model pollutant, the HCAOP process degraded 20 mg/L of dye in 5 L water within 2 min following a first-order reaction. The lowest electric energy per order (EEO) was 0.26 (kWh/m3/order). The HCAOP process is a highly efficient flow-type advanced oxidation process with potential industrial applications.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

Manipulating terahertz wave by a magnetically tunable liquid crystal phase grating.

This investigation demonstrates the feasibility of a magnetically tunable liquid crystal phase grating for the terahertz wave. The phase grating can be used as a beam splitter. The ratio of the zeroth and first-order diffracted THz-beams (0.3 THz) polarized in a direction perpendicular to that of the grooves of the grating can be tuned from 4:1 to 1:2. When the THz wave is polarized in any other direction, this device can be operated as a polarizing beam splitter.

Evaluation of effects from repeated inhalation exposure of F344 rats to high concentrations of propylene.

Chronic exposure to propylene does not result in any increased incidence of tumors, yet does increase N7-hydroxypropylguanine (N7-HPGua) adducts in tissue DNA. To investigate any potential for genotoxicity (mutagenicity or clastogenicity), male F344 rats were exposed via inhalation to up to 10,000 ppm propylene for 1, 3, or 20 days (6 h/day, 5 days/week). The endpoints examined included gene (Hprt, splenocytes) and chromosomal (bone marrow micronucleus [MN]) mutations, hemoglobin (hydroxypropylvaline, HPVal) adducts in systemic blood, and DNA adducts (N7-HPGua) in several tissues. Similarly exposed female and male F344 rats, implanted with bromodeoxyuridine (BrdU) minipumps, were evaluated for nasal effects (irritation via histopathology and cell proliferation via BrdU). Internal dose measures provided clear evidence for propylene exposure, with HPVal increased for all exposures; N7-HPGua was increased in all tissues from rats exposed for more than 1 day (except lymphocytes). Saturation of propylene conversion to propylene oxide was apparent from the adduct dose-response curves. There were no biologically significant genotoxic effects demonstrated at any exposure level, with no increase in Hprt mutant frequency or in bone marrow MN formation. In addition, no histopathological changes were noted in rodent nasal tissues nor any induction of cell proliferation in nasal tissues. These results demonstrate that repeated exposure of rats to high concentrations of propylene (< or = 10,000 ppm) does not produce evidence of local nasal cavity toxicity or evidence of systemic genotoxicity to hematopoietic tissue, despite the formation of N7-HPGua adducts. In addition, these data indicate that formation of N7-HPGua does not correlate with any measure of genotoxic effect, neither mutagenic nor clastogenic.

The importance of 3,4-epoxy-1,2-butanediol and hydroxymethylvinyl ketone in 3-butene-1,2-diol associated mutagenicity.

1,2:3,4-Diepoxybutane is hypothesized to be the main intermediate involved in mutagenicity following exposure to low levels of 1,3-butadiene (BD) in mice, while metabolites of 3-butene-1,2-diol (BD-diol) are thought to become involved in both rats and mice at higher exposures. BD-diol is biotransformed to hydroxymethylvinyl ketone (HMVK), a potentially mutagenic metabolite, and 3,4-epoxy-1,2-butanediol (EB-diol), a known mutagen. To determine the relative importance of HMVK and EB-diol in BD-diol associated mutagenesis, we have examined the dosimetry of a HMVK derived DNA adduct, as well as EB-diol derived DNA and hemoglobin adducts, in rodents exposed to BD-diol. We previously demonstrated similarities in the shapes of the dose-response curves for EB-diol derived DNA adducts, hemoglobin adducts, and Hprt mutant frequencies in BD-diol exposed rodents, indicating that EB-diol was involved in the mutagenic response associated with BD-diol exposure. To examine the role of HMVK in BD-diol mutagenicity, a method to quantify the alpha-regioisomer of HMVK derived 1,N(2)-propanodeoxyguanosine (alpha-HMVK-dGuo) was developed. The method involved enzymatic hydrolysis of DNA, HPLC purification, and adduct measurement by liquid chromatography - tandem mass spectrometry. Intra- and inter-experimental variabilities were determined to be 2.3-18.2 and 4.1%, respectively. The limit of detection was approximately 5 fmol of analyte standard injected onto the column or 5 fmol/200 microg DNA. The method was used to analyze liver DNA from control female F344 rats and female F344 rats exposed to 36 ppm BD-diol. In addition, liver samples from female Sprague-Dawley rats exposed to 1000 ppm BD were analyzed. alpha-HMVK-dGuo was not detected in any of the samples analyzed. Several possible explanations exist for the negative results including the possibility that alpha-HMVK-dGuo may be a minor adduct or may be efficiently repaired. Alternatively, HMVK itself may be readily detoxified by glutathione (GSH) conjugation. While experiments must be conducted to understand the exact mechanism(s), these results, in addition to published EB-diol derived adduct dosimetry and existing HMVK derived mercapturic acid data, suggest that EB-diol is primarily responsible for BD-diol induced mutagenicity in rodents.

Antibiotic-Induced Elevations of Plasma Bile Acids in Rats Independent of Bsep Inhibition.

Drug-induced liver injury (DILI) is a common toxicity observed in drug development and can lead to withdrawal of approved drugs from the market. To better understand the numerous mechanisms of DILI, recent efforts have focused on transporter inhibition, specifically liver canalicular bile salt export pump (Bsep) as one mechanism of DILI, and on the potential use of plasma bile acids as monitorable mechanism-based biomarkers of Bsep inhibition. To explore alternative mechanisms of bile acid increases in plasma, 6 antibiotic and 2 nonantibiotic drugs unlikely to be Bsep inhibitors were evaluated in rat studies. Surprisingly, all 6 antibiotics demonstrated 2- to 14-fold increases of plasma taurocholic acid (TCA). Also, unconjugated primary bile acids and secondary bile acids (both taurine-conjugated and unconjugated) were decreased in rat plasma after antibiotic treatments, but not with the nonantibiotic drugs. These results suggest alternative mechanisms of bile acids regulation such as attenuation of bacterial deconjugation of bile acids following reduction of gut microflora by antibiotics. Measurements of TCA transport in rat hepatocytes and Bsep-containing membrane vesicles suggest that inhibition of uptake into hepatocytes could also contribute to increases in plasma bile acid concentrations, while excluding inhibition of Bsep as a mechanism. These studies further demonstrate that there are several mechanisms that can lead to conjugated bile acid increases in plasma. By carefully considering the time course and magnitude of changes of individual bile acids relative to any changes seen in transaminases and bilirubin, interpretations and conclusions of the involvement of Bsep inhibition are enabled.

Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver.

Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8-oxoguanine DNA glycosylase, Mpg, AP endonuclease (Ape), polymerase beta (Pol beta) and alkylguanine methyltransferase were increased by 20-100% in livers of rats exposed to EO for 20 days. The only effects of ET on BER gene expression were observed in brain, where Ape and Pol beta expression were increased by less than 20% after 20 days of exposure to 3000 ppm. These data suggest that DNA damage induced by exposure to EO is repaired without accumulation of AP sites and is associated with biologically insignificant changes in BER gene expression in target organs. We conclude that accumulation of AP sites is not a likely primary mechanism for mutagenicity and carcinogenicity of EO.

Analysis of diepoxide-specific cyclic N-terminal globin adducts in mice and rats after inhalation exposure to 1,3-butadiene.

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane-specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)-valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers.

Spectral dependence of carrier lifetimes in silicon for photovoltaic applications.

Charge carrier lifetimes in photovoltaic-grade silicon wafers were measured by a spectral-dependent, quasi-steady-state photoconductance technique. Narrow bandwidth light emitting diodes (LEDs) were used to excite excess charge carriers within the material, and the effective lifetimes of these carriers were measured as a function of wavelength and intensity. The dependence of the effective lifetime on the excitation wavelength was then analyzed within the context of an analytical model relating effective lifetime to the bulk lifetime and surface recombination velocity of the material. The agreement between the model and the experimental data provides validation for this technique to be used at various stages of the solar cell production line to investigate the quality of the passivation layers and the bulk properties of the material.

Intracavity measurement of liquid crystal layer thickness by wavelength tuning of an external cavity laser diode.

The gap of a planar-aligned liquid crystal (LC) cell is measured by a novel method: Monitoring the change in output wavelength of an external-cavity diode laser by varying the voltage driving the LC cell placed in the laser cavity. This method is particularly suitable for measurement of LC cells of small phase retardation. Measurement errors of +/-0.5 % and +/-0.6 % for 9.6-microm and 4.25-microm cells with phase retardations of 1.63 microm and 0.20 microm respectively are demonstrated.

Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis.

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied (dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade (shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom (phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase (Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3beta,5beta-ketodiol) conversion to 2,22-dideoxyecdysone (3beta,5beta-ketotriol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval-larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme.

Comparison of continuous-wave terahertz wave generation and bias-field-dependent saturation in GaAs:O and LT-GaAs antennas.

Terahertz wave (THz) photoconductive (PC) antennas were fabricated on oxygen-implanted GaAs (GaAs:O) and low-temperature-grown GaAs (LT-GaAs). The measured cw THz power at 0.358 THz from the GaAs:O antenna is about twice that from the LT-GaAs antenna under the same testing conditions, with the former showing no saturation up to a bias of 40 kV/cm, while the latter is already beginning to saturate at 20 kV/cm. A modified theoretical model incorporating bias-field-dependent electron saturation velocity is employed to explain the results. It shows that GaAs:O exhibits a higher electron saturation velocity, which may be further exploited to generate even larger THz powers by reducing the ion dosage and optimizing the annealing process in GaAs:O.

Investigation on spectral loss characteristics of subwavelength terahertz fibers.

By measuring the spectral loss characteristics of subwavelength-diameter terahertz fibers, our study supports the recent theory proposed by M. Sumetsky [Opt. Lett. 31, 870 (2006)] that diameter-variation-induced radiation is a dominant loss mechanism for subwavelength fibers in the low- (<1%) core-fraction-power regime. This physical mechanism limits the lowest guidable frequency in a subwavelength fiber.

Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry.

A sensitive method using high-performance liquid chromatography coupled to a mass spectrometer with electrospray ionization source (HPLC/ESI-MS) was developed for detection of ecdysteroids in biological samples. We report here for the first time that ecdysteroids can be classified into three groups based on ESI full-scan mass spectra: group 1 (ecdysone (E), 2-deoxyecdysone (2dE), 2,22-dideoxyecdysone (3beta5beta-KT), and 3alpha5alpha[H]-dihydroxycholest-7-en-6-one (3alpha5alpha-KD)), in which loss of one molecule of water from the protonated molecular ion ([M+H](+)) represents the dominant ion; group 2 (20-hydroxyecdysone (20E), makisterone A (MakA), 3beta5beta-KD, and 3beta5alpha-KD), in which [M+H](+) is a major ion but some water loss is observed; and group 3 (muristerone A (MurA) and ponasterone A (PonA)), in which [M+H](+) is the dominant ion with no water loss observed. Based on the analytical procedure in combination with structural information from the group classification and with the application of source-induced dissociation, we identified free ecdysteroids in biological samples: 20,26-dihydroxyecdysone and ecdysonic acid in the larval hemolymph, and the progressive metabolism of 26-hydroxyecdysone (26E) to 3alpha-26E from day-1 to day-3 embryos of the tobacco hornworm Manduca sexta.

LC/MS/MS method for the quantitation of trans-2-hexenal-derived exocyclic 1,N(2)-propanodeoxyguanosine in DNA.

trans-2-Hexenal is an alpha,beta-unsaturated aldehyde to which humans are exposed daily in small amounts. Hexenal has demonstrated mutagenicity and genotoxicity in vitro and reacts with deoxyguanosine to form diastereomeric hexenal-derived exocyclic 1,N(2)-propanodeoxyguanosine (Hex-PdG) adducts. A highly sensitive and specific method for the measurement of Hex-PdG in DNA has not previously been available. An LC/MS/MS assay for the quantitation of Hex-PdG, using [(13)C4(15)N2]Hex-PdG as an internal standard, was developed, to assess binding of hexenal to DNA. Samples were purified prior to analysis by centrifuge filtration and solid phase extraction and analyzed by LC/MS/MS in the selected reaction monitoring (SRM) mode (SRM m/z 366.2 --> 250.2 for Hex-PdG; SRM m/z 372.2 --> 256.2 for [(13)C4(15)N2]Hex-PdG). Recovery of standards was 89% or greater, and quantitation was unaffected by the addition of increasing concentrations of calf thymus DNA (ctDNA). The limit of quantitation, determined in samples of 200 microg of ctDNA spiked with analyte standard, was 0.015 fmol/microg DNA, which corresponds to approximately 5 Hex-PdG/10(9) unmodified nucleotides. Hex-PdG was detected in ctDNA treated with 0.021 microM, 0.21 microM, or 2.1 mM hexenal but not in untreated DNA. Furthermore, Hex-PdG was not detected in DNA exposed to reactive oxygen species-mediated deoxyribose attack and lipid peroxidation, which resulted in a significant increase in the malondialdehyde-derived pyrimido[1,2-a]purin-10(3H)one. Hex-PdG was not detected in DNA of untreated rat liver, but Hex-PdG in hexenal-treated calf thymus DNA was quantifiable when spiked into the rat liver DNA at 0.035 or 0.35 fmol/microg DNA. These data indicate that Hex-PdG is formed following hexenal treatment and that this method is suitable for in vitro or in vivo assessment of Hex-PdG formation.

Anti-AIDS agents 65: investigation of the in vitro oxidative metabolism of 3',4'-Di-O-(-)-camphanoyl-(+)-cis-khellactone derivatives as potent anti-hiv agents.

3',4'-Di-O-(-)-camphanoyl-(+)-cis-khellactone (DCK) is a synthetic khellactone ester that exhibits potent in vitro anti-human immunodeficiency virus (HIV) activity with a mechanism distinct from clinically used anti-HIV agents. Several series of mono- and di-substituted DCK derivatives (DCKs) have previously been synthesized, and their structure-activity relationships are well established. To optimize DCK as a drug lead and to guide further structural modifications, metabolic stabilities and metabolite structures were analyzed. In vitro metabolic stabilities of DCKs in human liver microsomes were assessed using high performance liquid chromatography (HPLC) with UV detection to establish structure-metabolism relationships (SMRs). HPLC coupled with ion trap mass spectrometry was used to identify the metabolite structures. The results indicated that DCKs undergo rapid oxidation on the lipophilic camphanoyl moieties and the substituents on the khellactone do not alter the rate or the metabolic pathways for this compound type. Our SMR and metabolite analysis study suggested that the two camphanoyl ester moieties are the determinants of the low metabolic stability and that structural alteration in the two esters may be necessary to improve metabolic profiles of DCKs.

Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone.

The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.