Feasibility of wearable technology for 'real-world' gait analysis in children with Prader-Willi and Angelman syndromes.

BACKGROUND: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders in need of innovative 'real-world' outcome measures to evaluate treatment effects. Instrumented gait analysis (IGA) using wearable technology offers a potentially feasible solution to measure "real-world' neurological and motor dysfunction in these groups. METHODS: Children (50% female; 6-16 years) diagnosed with PWS (n = 9) and AS (n = 5) completed 'real-world' IGA assessments using the Physilog®5 wearable. PWS participants completed a laboratory assessment and a 'real-world' long walk. The AS group completed 'real-world' caregiver-assisted assessments. Mean and variability results for stride time, cadence, stance percentage (%) and stride length were extracted and compared across three different data reduction protocols. RESULTS: The wearables approach was found to be feasible, with all participants able to complete at least one assessment. This study also demonstrated significant agreement, using Lin's concordance correlation coefficient (CCC), between laboratory and 'real-world' assessments in the PWS group for mean stride length, mean stance % and stance % CV (n = 7, CCC: 0.782-0.847, P = 0.011-0.009). CONCLUSION: 'Real-world' gait analysis using the Physilog®5 wearable was feasible to efficiently assess neurological and motor dysfunction in children affected with PWS and AS.

Barriers and facilitators to smoking cessation among HIV-infected people who inject drugs (PWID) in Hanoi, Vietnam: a qualitative study.

INTRODUCTION: In Vietnam, 60% of men living with HIV smoke tobacco, and 92% of HIV-infected people who inject drugs (PWID) smoke tobacco. Tobacco use increases mortality through increased health risks including tuberculosis and malignancy in HIV-infected smokers. However, tobacco use treatment is not widely available in Vietnam. The objective was to examine current barriers and facilitators of smoking cessation and tobacco use treatment for HIV-infected PWID in Hanoi, Vietnam. METHODS: Native speaking ethnographers conducted semi-structured qualitative interviews about tobacco use and tobacco use treatment with sixteen HIV-infected PWID and eight healthcare providers, recruited from four HIV-Methadone Maintenance Treatment (MMT) clinics in Hanoi, Vietnam. Interviews were recorded, transcribed, and translated for thematic analysis in Dedoose. RESULTS: Clients and providers had learned the general health risks of smoking from public awareness campaigns. Half had tried to quit previously, often motivated by advice from family members but not by HIV providers' advice. Almost all clients did not want to quit, citing the low price of tobacco, prevalence of smoking in Vietnam, and physical cravings. HIV provider's counseling was brief, inconsistent, and limited by low provider knowledge and competing burdens of HIV and injection drug use. Providers recently trained by NGO-led seminars on tobacco prioritized tobacco use treatment. CONCLUSIONS: Smoking cessation efforts for people living with HIV/AIDS (PLHA) and PWID smokers in Hanoi, Vietnam could benefit from further community public awareness campaigns, and exploring increased tobacco taxation. Tobacco use treatment at HIV clinics could benefit from involving family and friends in cessation, and training providers in treatment methods.

Determination of Growth-Phase Dependent Influences Exerted by Prions on Yeast Lipid Content Using HPTLC-Densitometry.

Prions of the baker's yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function(s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI+], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)-densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane-petroleum ether-diethyl ether-acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5), cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([PSI+] or [URE3]) and prion-free ([psi-] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC-densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.

Controllable structural and optical properties of NaYF4:Tm, Yb microparticles by Yb3+ doping for anti-counterfeiting.

Hexagonal NaYF4:Tm, Yb upconversion (UC) phosphors with excellent UC luminescence quantum efficiency and chemical stability meet demands for applications in bioimaging and anti-counterfeiting printing. In this work, a series of NaYF4:Tm, Yb upconversion microparticles (UCMPs) with different concentrations of Yb were synthesized by a hydrothermal method. Then, the UCMPs become hydrophilic through surface oxidation of the oleic acid (C-18) ligand to azelaic acid (C-9) using the Lemieux-von Rodloff reagent. The structure and morphology of UCMPs were investigated by X-ray diffraction and scanning electron microscopy. The optical properties were studied using diffusion reflectance spectroscopy and photoluminescent spectroscopy under 980 nm laser irradiation. The emission peaks of the Tm3+ ions are 450, 474, 650, 690, and 800 nm, attributed to the transitions from the excited state to ground state 3H6. These emissions are the results of two or three photon absorption through multi-step resonance energy transfer from excited Yb3+, confirmed via a power-dependent luminescence study. The results show that the crystal phases and luminescence properties of the NaYF4:Tm, Yb UCMPs are controlled by changing the Yb doping concentration. The printed patterns are readable under the excitation of a 980 nm LED. Moreover, the zeta potential analysis shows that the UCMPs after surface oxidation are water dispersible. In particular, the naked eye can observe the enormous upconversion emissions in UCMPs. These findings indicated that this fluorescent material is an ideal candidate for anti-counterfeiting and biological applications.

Effect of gold immersion time on the electrochemical migration property of electroless nickel/immersion gold surface finishing.

In this study, the electrochemical performance of an electroless nickel/immersion gold (ENIG) surface finish was evaluated as a function of the Au immersion time by the water immersion migration test. As the Au plating time increased, the electroless nickel phosphorous (EN-P) changed from amorphous to crystalline and then increased in crystallinity. X-ray diffraction (XRD) was used to evaluate the crystallinity of the plating layer. The electrical resistance of the electrodes was tracked as the sample was immersed in water with a 5 V bias. The microstructures of the electrodes after the electrochemical migration test were observed by using secondary electron microscopy (SEM) and energy dispersive spectroscopy (EDS). As the Au immersion time increased, the EN-P's crystallinity and Au thickness increased. This enhanced the electrochemical migration protection of the surface finish layer.

"I refused to drink but they still forced me": A mixed-methods approach to understanding the pathways to reduce alcohol use among Vietnamese people with HIV.

AIMS: This study explores the effects of two evidence-based alcohol reduction counseling interventions on readiness to change, alcohol abstinence self-efficacy, social support, and alcohol abstinence stigma among people with HIV (PWH) who have hazardous alcohol use in Vietnam. METHODS: PWH receiving antiretroviral therapy (ART) were screened for hazardous drinking and randomized to one of three study arms: combined intervention (CoI), brief intervention (BI), and standard of care (SOC). A quantitative survey was conducted at baseline (N = 440) and 3-month post-intervention (N = 405), while in-depth interviews were conducted with a subset of BI and CoI participants at baseline (N = 14) and 3 months (N = 14). Data was collected from March 2016 to August 2017. A concurrent mixed-methods model was used to triangulate quantitative and qualitative data to cross-validate findings. RESULTS: At 3 months, receiving the BI and CoI arms was associated with 2.64 and 3.50 points higher in mean readiness to change scores, respectively, compared to the SOC group (BI: ß = 2.64, 95% CI: 1.17-4.12; CoI: ß = 3.50, 95% CI 2.02-4.98). Mean alcohol abstinence self-efficacy scores were 4.03 and 3.93 points higher among the BI and CoI arm at 3 months, compared to SOC (BI: ß = 4.03, 95% CI: 0.17-7.89; CoI: ß = 3.93, 95% CI: 0.05-7.81). The impacts of the interventions on social support and alcohol abstinence stigma were not significant. Perceived challenges to refusing drinks at social events remained due to strong alcohol abstinence stigma and perceived negative support from family and friends who encouraged participants to drink posed additional barriers to reducing alcohol use. CONCLUSIONS: Both the CoI and BI were effective in improving readiness to change and alcohol abstinence self-efficacy among PWH. Yet, participants still faced significant barriers to reducing their drinking due to social influences and pressure to drink. Interventions at different levels addressing social support and alcohol abstinence stigma are warranted.

Mobilization of retrotransposons in synthetic allotetraploid tobacco.

Allopolyploidy is a major driving force in plant evolution and can induce rapid structural changes in the hybrid genome. As major components of plant genomes, transposable elements are involved in these changes. In a previous work, we observed turnover of retrotransposon insertions in natural allotretraploid tobacco (Nicotiana tabacum). Here, we studied the early stages of allopolyploid formation by monitoring changes at retrotransposon insertion sites in the Th37 synthetic tobacco. We used sequence-specific amplification polymorphism (SSAP) to study insertion patterns of two populations of the Tnt1 retrotransposon in Th37 S4 generation plants, and characterized the nature of polymorphic insertion sites. We observed significant amplification of young Tnt1 populations. Newly transposed copies were amplified from maternal elements and were highly similar to Tnt1A tobacco copies amplified in response to microbial factors. A high proportion of paternal SSAP bands were not transmitted to the hybrid, corresponding to various rearrangements at paternal insertion sites, including indels or the complete loss of the Tnt1/flanking junction. These data indicate that major changes, such as retrotransposon amplification and molecular restructuring in or around insertion sites, occur rapidly in response to allopolyploidy.

Monitoring for Plasmodium falciparum drug resistance to artemisinin and artesunate in Binh Phuoc Province, Vietnam: 1998-2009.

BACKGROUND: Artemisinin derivatives have been used for malaria treatment in Vietnam since 1989. Reported malaria cases have decreased from 1,672,000 with 4,650 deaths in 1991, to 91,635 with 43 deaths in 2006. Current national guidelines recommend artemisinin-based combination therapy (ACT), although artesunate is still available as monotherapy through the private sector. Recent reports suggest that effectiveness of ACT and artesunate monotherapy has declined in western Cambodia. This study examined Plasmodium falciparum resistance patterns over 10 years in southwest Vietnam in infected patients treated with artemisinin compounds. METHODS: The study was conducted in two communes in Phuoc Long district, Binh Phuoc province, 100 km west of the Cambodian border. This was chosen as a likely site for emerging artemisinin resistance because of the high prevalence of P. falciparum malaria, and the length of time that artemisinin had been in use. In vivo and in vitro monitoring of P. falciparum susceptibility to anti-malarial drugs was conducted in 1998, 2001, 2004/5, and 2008/9. Patients with confirmed P. falciparum malaria received therapy with 5 or 7 days of artemisinin (1998 and 2001 respectively) or 7 days of artesunate RESULTS: In the four surveys, 270 patients were recruited and treated. The mean parasite clearance times differed between 1998, 2001 and 2004/5 (1.8, 2.3 and 2.1 days, P < 0.01) but not between 1998 and 2008/2009. The mean parasite clearance times were correlated with parasite density at day 0 (r = 0.4; P < 0.001). Treatment failure rates after PCR adjustment were 13.8%, 2.9%, 1.2%, and 0% respectively. Susceptibility of P. falciparum to artemisinin in in vitro tests was stable during the period, except for a rise in EC90 and EC99 in 2001. CONCLUSIONS: This study showed stable levels of P. falciparum sensitivity to artemisinin compounds in the two sites over a ten-year period. The introduction of ACT in this area in 2003 may have protected against the development of artemisinin resistance. Adherence to the latest WHO and Vietnamese guidelines, which recommend ACT as first-line therapy in all malarious areas, and continued monitoring along the Vietnam-Cambodia border will be essential to prevent the spread of artemisinin resistance in Vietnam.

Social support modifies the association between hazardous drinking and depression symptoms among ART clients in Vietnam.

INTRODUCTION: Hazardous drinking is widespread among people with HIV (PWH). PWH are also vulnerable to depression due to HIV-related social stigma, and social support can play an important role in improving mental health for this population. No studies have explored whether social support modifies the association of hazardous drinking and depressive symptoms among PWH. METHODS: We used baseline data from a randomized controlled trial of two evidence-based alcohol reduction interventions among antiretroviral therapy clients in Vietnam. Hazardous alcohol use was defined as having a score ≥8 for men and ≥ 7 for women on the Alcohol Use Disorders Identification Test. The presence of depression symptoms was defined as a score ≥ 5 on the Patient Health Questionnaire-9. Social support was measured with a 5-question modified version of the Medical Outcomes Study Social Support Instrument. Crude (CPRs) and adjusted prevalence ratios (aPRs) of the association were presented. RESULTS: Hazardous drinking was significantly associated with increased likelihood of having depressive symptoms (aPR = 1.26;95%CI 1.04-1.52). Hazardous drinking and depression symptoms were not associated among those with high social support (aPR = 1.01;95%CI 0.76-1.35), but were associated among those with medium (aPR = 1.24;95%CI 0.92-1.69) and low social support (aPR = 1.71;95%CI 1.25-2.34). CONCLUSIONS: Social support significantly modified the association between hazardous drinking and depression symptoms among ART clients in Vietnam. Interventions to decrease hazardous alcohol use are broadly indicated for PWH in Vietnam and other low-resource settings, but special attention or modifications may be needed to support mental health among those with lower levels of social support.

Small CGG repeat expansion alleles of FMR1 gene are associated with parkinsonism.

Fragile X-associated tremor/ataxia syndrome (FXTAS) affects older males carrying premutation, that is, expansions of the CGG repeat (in the 55-200 range), in the FMR1 gene. The neurological changes are linked to the excessive FMR1 messenger RNA (mRNA), becoming toxic through a 'gain-of-function'. Because elevated levels of this mRNA are also found in carriers of the smaller expansion (grey zone) alleles, ranging from 40 to 54 CGGs, we tested for a possible role of these alleles in the origin of movement disorders associated with tremor. We screened 228 Australian males affected with idiopathic Parkinson's disease and other causes of parkinsonism recruited from Victoria and Tasmania for premutation and grey zone alleles. The frequencies of either of these alleles were compared with the frequencies in a population-based sample of 578 Guthrie spots from consecutive Tasmanian male newborns (controls). There was a significant excess of premutation carriers (Fisher's exact test p = 0.006). There was also a more than twofold increase in grey zone carriers in the combined sample of the Victorian and Tasmanian cases, with odds ratio (OR ) = 2.36, and 95% confidence intervals (CI): 1.20-4.63, as well as in Tasmanian cases only (OR = 2.33, 95% CI: 1.06-5.13), compared with controls. The results suggest that the FMR1 grey zone alleles, as well as premutation alleles, might contribute to the aetiology of disorders associated with parkinsonism.

Novel urchin-like FeCo oxide nanostructures supported carbon spheres as a highly sensitive sensor for hydrazine sensing application.

Herein, we successfully fabricated a novel nanostructure based on hierarchical urchin-like FeCo oxide supported carbon spheres (FeCo Oxide/CSs) via a two-step hydrothermal method followed by a simple annealing step at 300 °C under air. It was found that such urchin-like FeCo Oxide/CSs structure exhibited superior catalytic activity towards hydrazine oxidation to CSs, Fe Oxide/CSs, Co Oxide/CSs, and FeCo Hydroxide/CSs material. In this regard, the FeCo Oxide/CSs displayed a wide linear detection range of 0.1-516.6 µM, low detection limit of 0.1 µM, and long-term stability. The material also showed good selectivity towards hydrazine detection in the presence of various interferences, such as uric acid, ascorbic acid, urea, dopamine, Na+, SO42-, K+, and Cl-. The excellent sensing performance of the FeCo Oxide/CSs was assumed to the unique hierarchical urchin structure with the high density and uniformity of nano-sized FeCo Oxide nanoneedles, which produced massive electroactive sites and enhanced charge transfer ability. The achieved results implied that the FeCo Oxide/CSs may be a great candidate for sensitive hydrazine detection.

Prevalence of polymorphisms in DHFR, DHPS, PFMDR1 and PFCRT genes of Plasmodium falciparum isolates in Quang Tri Province, Vietnam.

In 2002 an antimalarial drug resistance survey was carried out in a seasonally endemic area of Vietnam. Sulfadoxine/pyrimethamine (S/P) was the standard treatment recommended for uncomplicated Plasmodium falciparum malaria in that area at the time. Early or late treatment failure as defined by WHO was observed in 14.9% (7/47) of patients. Molecular analysis of treatment failure isolates identified that 5/6 carried two or more dhfr and dhps polymorphisms associated with S/P resistance. Chloroquine resistance-associated polymorphisms occurred in 38.5% (15/39) of the isolates. These results support the move to artemisinin-based combination therapy for malaria in Vietnam.

ß-glucuronidase mRNA levels are correlated with gait and working memory in premutation females: understanding the role of FMR1 premutation alleles.

Fragile X tremor ataxia syndrome (FXTAS) is a late-onset disorder manifesting in a proportion of FMR1 premutation individuals (PM: 55-199 CGG triplet expansions). FXTAS is associated with elevated levels of FMR1 mRNA which are toxic. In this study, relationships between neurocognitive and intra-step gait variability measures with mRNA levels, measured in blood samples, were examined in 35 PM and 35 matched control females. The real-time PCR assays measured FMR1 mRNA, and previously used internal control genes: ß-Glucuronidase (GUS), Succinate Dehydrogenase 1 (SDHA) and Eukaryotic Translation Initiation Factor 4A (EI4A2). Although there was significant correlation of gait variability with FMR1 mRNA levels (p = 0.004) when normalized to GUS (FMR1/GUS), this was lost when FMR1 was normalized to SDHA and EI4A2 (2IC). In contrast, GUS mRNA level normalized to 2IC showed a strong correlation with gait variability measures (p < 0.007), working memory (p = 0.001) and verbal intelligence scores (p = 0.008). PM specific changes in GUS mRNA were not mediated by FMR1 mRNA. These results raise interest in the role of GUS in PM related disorders and emphasise the importance of using appropriate internal control genes, which have no significant association with PM phenotype, to normalize FMR1 mRNA levels.

Brain structure and intragenic DNA methylation are correlated, and predict executive dysfunction in fragile X premutation females.

DNA methylation of the Fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary has been associated with executive dysfunction in female carriers of a FMR1 premutation (PM: 55-199 CGG repeats), whereas neuroanatomical changes have been associated with executive dysfunction in PM males. To our knowledge, this study for the first time examined the inter-relationships between executive function, neuroanatomical structure and molecular measures (DNA methylation and FMR1 mRNA levels in blood) in PM and control (<44 CGG repeats) females. In the PM group, FMR1 intron 1 methylation was positively associated with executive function and cortical thickness in middle and superior frontal gyri, and left inferior parietal gyrus. By contrast, in the control group, FMR1 intron 1 methylation was negatively associated with cortical thickness of the left middle frontal gyrus and superior frontal gyri. No significant associations were revealed for either group between FMR1 mRNA and neuroanatomical structure or executive function. In the PM group, the lack of any significant association between FMR1 mRNA levels and phenotypic measures found in this study suggests that either FMR1 expression is not well conserved between tissues, or that FMR1 intron 1 methylation is linked to neuroanatomical and cognitive phenotype in PM females via a different mechanism.

Molecular genetic analysis of Drosophila eyes absent mutants reveals an eye enhancer element.

The eyes absent (eya) gene is critical for normal eye development in Drosophila and is highly conserved to vertebrates. To define regions of the gene critical for eye function, we have defined the mutations in the four viable eya alleles. Two of these mutations are eye specific and undergo transvection with other mutations in the gene. These were found to be deletion mutations that remove regulatory sequence critical for eye cell expression of the gene. Two other viable alleles cause a reduced eye phenotype and affect the function of the gene in additional tissues, such as the ocelli. These mutations were found to be insertion mutations of different transposable elements within the 5' UTR of the transcript. Detailed analysis of one of these revealed that the transposable element has become subject to regulation by eye enhancer sequences of the eya gene, disrupting normal expression of EYA in the eye. More extended analysis of the deletion region in the eye-specific alleles indicated that the deleted region defines an enhancer that activates gene expression in eye progenitor cells. This enhancer is responsive to ectopic expression of the eyeless gene. This analysis has defined a critical regulatory region required for proper eye expression of the eya gene.

Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain.

The eyes absent (eya) gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other genes of eye development, sine oculis and dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, we have sequenced eya alleles. These mutations have revealed that loss of the entire Eya Domain is null for eya activity, but that alleles with truncations within the Eya Domain display partial function. We then extended the molecular genetic analysis to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminished the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it.

Monooxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase.

Microsomes from rat anterior pituitaries (AP) were incubated with (3H)estradiol under conditions previously shown to support catecholestrogen (CE) formation by placental microsomes via an NADPH- or an organic hydroperoxide-dependent, peroxidatic mechanism. Under conditions optimized for monooxygenase activity (pH 8.0, 5 mM NADPH), 4-hydroxylation predominated (apparent Vmax = 65 pmol and 13 pmol/mg protein/30 min for 4- and 2-hydroxy-E2, respectively). Under conditions optimized for peroxidatic activity (pH 6.0, 50 mM cumene hydroperoxide) 2- and 4-hydroxylated-E2 were produced in similar amounts. Thus in the AP, unlike in other target tissues studied, NADPH-dependent CE synthetase is a 4-hydroxylase and significant 2-hydroxylation occurs only via the peroxidatic mechanism. We propose that 4-hydroxylated CEs, which are both potent, long acting estrogens and catechols, serve as local mediators of actions of phenolic estrogens on the AP.

Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens.

Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.

Conversion of estrone to 2- and 4-hydroxyestrone by hamster kidney and liver microsomes: implications for the mechanism of estrogen-induced carcinogenesis.

As part of an ongoing investigation of the role of metabolic activation of estrogens in the genesis of cancers such as estrogen-induced renal tumors in hamsters, we have 1) determined steroid-17 beta-oxidoreductase activity of microsomes and cytosol prepared from hamster kidney and liver; 2) compared the rates of 2-, 4-, and 16 alpha-hydroxylations of estrone by microsomes from hamster kidney and liver; and 3) determined the rates of inactivation of 2- and 4-hydroxyestrone by catechol-O-methyltransferase from hamster kidney and by purified enzyme. Microsomal steroid-17 beta-oxidoreductase activity in hamster kidney and liver was low and favored the conversion of estrone to estradiol. Cytosolic steroid-17 beta-oxidoreductase activity was only barely detectable in both liver and kidney. Using hepatic microsomes, the rate of 2-hydroxylation of estrone was comparable to that found previously using estradiol as substrate, whereas 4-hydroxylation of estrone was double that of estradiol. Using renal microsomes, the rates of 2- and 4-hydroxylation of estrone were 10- to 20-fold higher than those with estradiol as substrate, and the ratio of 2- to 4-hydroxylation was about 2:1. Fadrozole hydrochloride was an equally good inhibitor of rates of 2- and 4-hydroxylation of estrone (20 microM) by hepatic microsomes (IC50, approximately 25 microM). Corresponding IC50 values with renal microsomes were less than 2 microM, and 2-hydroxylation of estrone was inhibited by Fadrozole hydrochloride up to 15% more than 4-hydroxylation. Treatment of hamsters with estradiol for 2 months decreased rates of 2- and 4-hydroxylation of estrone by renal microsomes by approximately 95%. The rate of conversion of estrone to 16 alpha-hydroxyestrone by hepatic microsomes was 10-20% that of 2-hydroxylation. Renal microsomes catalyzed 16 alpha-hydroxylation of estrone at an even lower rate (approximately 5% of that of 2-hydroxylation). Rates of O-methylation of 2- and 4-hydroxyestrone by hamster kidney cytosol were comparable to those of 2- and 4-hydroxyestradiol. In conclusion, conversion of estrone to its catechol metabolites by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, is quantitatively more important than the conversion to 16 alpha-hydroxyestrone. The findings are consistent with the postulated role of catechol estrogens generated in situ in estrone-induced carcinogenesis.

Hepatic catecholestrogen synthases: differential effect of sex, inducers of cytochromes P-450 and of antibody to the glucocorticoid inducible cytochrome P-450 on NADPH-dependent estrogen-2-hydroxylase and on organic hydroperoxide-dependent estrogen-2/4-hydroxylase activity of rat hepatic microsomes.

Formation of catecholestrogens (CE) by rat hepatic microsomes was re-examined because as recently shown; (1) CE formation can be catalyzed by an NADPH-dependent estrogen-4-hydroxylase (E-4-H(NADPH)) and by a peroxidatic, organic hydroperoxide-dependent estrogen-2/4-hydroxylase (E-2/4-H(OHP)), in addition to the established NADPH-dependent estrogen 2-hydroxylase (E-2-H(NADPH)); and (2) the indirect radiometric and the COMT-coupled radioenzymatic assays, used in many previous studies, may fail to provide an accurate measure, in particular, of 4-OH-CE. Using a direct product isolation assay, hepatic microsomes of both male and female rats were shown to express E-2/4-H(OHP) activity with properties similar to those of peroxidatic activity in other tissues. The activities of E-2/4-H(OHP) and E-2-H(NADPH) were affected differently by 5 out of 7 inducers of cytochromes P-450 administered in vivo. Phenobarbital and dexamethasone caused a 4- and 2-3-fold increase in E-2-H(NADPH) activity, respectively, but only a 38 and 20% increase in E-2/4-H(OHP) activity. Ketoconazol and beta-naphtoflavone caused a modest increase in E-2-H(NADPH) activity but a decrease in OHP-dependent activity. Clofibrate decreased peroxidatic activity by 50% and NADPH-dependent activity by approximately 20%. Both activities were increased by ethanol but decreased by isoniazide, an agent which induces the same form of cytochromes P-450 as ethanol. Polyclonal antibody against P-450p, a form of P-450 induced by glucocorticoids, inhibited E-2-H(NADPH) but not E-2/4-H(OHP) activity of untreated and of dexamethasone- and phenobarbital-treated rats. This study establishes that CE formation may occur in liver via the peroxidatic pathway and indicates that this pathway depends on forms of P-450 different from those mediating E-2-H(NADPH) activity. It also confirms and extends previous observations of the involvement of multiple, constitutive and induced forms of cytochrome P-450 in NADPH-dependent 2-hydroxylation in liver.