Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

The effectiveness of a needleless intravenous system in prevention of percutaneous injury in two hospitals

This study assessed if hospital-wide implementation of a needleless intravenous connection system reduces the number of reported percutaneous injuries, overall and those specifically due to intravenous connection activities.^ Incidence rates were compared before and after hospital-wide implementation of a needleless intravenous system at two hospitals, a full service general hospital and a pediatric hospital. The years 1989-1991 were designated as pre-implementation and 1993 was designated as post-implementation. Data from 1992 were not included in the effectiveness evaluation to allow employees to become familiar with use of the new device. The two hospitals showed rate ratios of 1.37 (95% CI = 1.22-1.54, p $\le$.0001) and 1.63 (95% CI = 1.34-1.97, p $\le$.0001), or a 27.1% and a 38.6% reduction in overall injury rate, respectively. Rate ratios for intravenous connection injuries were 2.67 (95% CI = 1.89-3.78, p $\le$.0001) and 3.35 (95% CI = 1.87-6.02, p $\le$.0001), or a 62.5% and a 69.9% reduction in injury rate, respectively. Rate ratios for all non-intravenous connection injuries were calculated to control for factors other than device implementation that may have been operating to reduce the injury rate. These rate ratios were lower, 1.21 and 1.44, demonstrating the magnitude of injury reduction due to factors other than device implementation. It was concluded that the device was effective in reduction of numbers of reported percutaneous injuries.^ Use-effectiveness of the system was also assessed by a survey of randomly selected device users to determine satisfaction with the device, frequency of use and barriers to use. Four hundred seventy-eight surveys were returned for a response rate of 50.9%. Approximately 94% of respondents at both hospitals expressed satisfaction with the needleless system and recommended continued use. The survey also revealed that even though over 50% of respondents report using the device "always" or "most of the time" for intravenous medication administration, flushing lines, and connecting secondary intravenous lines, needles were still being used for these same activities. Compatibility, accessibility and other technical problems were reported as reasons for using needles for these activities. These problems must be addressed, by both manufacturers and users, before the needleless system will be effective in prevention of all intravenous connection injuries. ^

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH- cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs, and pollutants. The functions of this system are as diverse as the substrates. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. This enzyme system has the highest level of activity in the liver. It is also present in other tissues, including brain. The functions of this enzyme system in brain seem to include: neurotransmission, neuroendocrinology, developmental and behavioral modulation, regulation of intracellular levels of cholesterol, and potential neurotoxicity.^ In this study, we have set up the rat glioma C6 cell line as an in vitro model system to examine the expression, induction, and tissue-specific regulation of P450s and P450 reductase. Rat glioma C6 cells were treated with P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). The presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction digestion. The induction of P450 1A1 and 2B1/2 and P450 reductase was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P450 1A, 2B and P450 reductase at the protein level. ELISAs showed that BA and PB induce P450 1A and 2B proteins 7.3- and 13.5-fold, respectively. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 CO difference spectra with absorption at or near 450 nm. Microsomes prepared from rat glioma C6 cells demonstrated much higher levels of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activity, when treated with BA or PB, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system which can be induced by P450 inducers. The mRNAs of P450 1A1 and 2B1/2 can not bind to the oligo(dT) column efficiently, indicating they have very short poly(A) tails. This finding leads us to study the tissue specific regulation of P450s at post-transcriptional level. The half lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are only 1/10 and 1/3 of that in liver. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors. ^

The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

Racial and ethnic disparities in diabetes mellitus and hypertension: Influence of income and insurance status in a cross-sectional study of the United States

Racial/ethnic disparities in diabetes mellitus (DM) and hypertension (HTN) have been observed and explained by socioeconomic status (education level, income level, etc.), screening, early diagnosis, treatment, prognostic factors, and adherence to treatment regimens. To the author's knowledge, there are no studies addressing disparities in hypertension and diabetes mellitus utilizing Hispanics as the reference racial/ethnic group and adjusting for sociodemographics and prognostic factors. This present study examined racial/ethnic disparities in HTN and DM and assessed whether this disparity is explained by sociodemographics. To assess these associations, the study utilized a cross-sectional design and examined the distribution of the covariates for racial/ethnic group differences, using the Pearson Chi Square statistic. The study focused on Non-Hispanic Blacks since this ethnic group is associated with the worst health outcomes. Logistic regression was used to estimate the prevalence odds ratio (POR) and to adjust for the confounding effects of the covariates. Results indicated that except for insurance coverage, there were statistically significant differences between Non-Hispanic Blacks and Non-Hispanic Whites, as well as Hispanics with respect to study covariates. In the unadjusted logistic regression model, there was a statistically significant increased prevalence of hypertension among Non-Hispanic Blacks compared to Hispanics, POR 1.36, 95% CI 1.02-1.80. Low income was statistically significantly associated with increased prevalence of hypertension, POR 0.38, 95% CI 0.32-0.46. Insurance coverage, though not statistically significant, was associated with an increase in the prevalence of hypertension, p>0.05. Concerning DM, Non-Hispanic Blacks were more likely to be diabetic, POR 1.10, 95% CI 0.85-1.47. High income was statistically significantly associated with decreased prevalence of DM, POR 0.47, 95% CI 0.39-0.57. After adjustment for the relevant covariates, the racial disparities between Hispanics and Non-Hispanic Blacks in HTN was removed, adjusted prevalence odds (APOR) 1.21, 95% CI 0.88-1.67. In this sample, there was racial/ethnic disparity in hypertension but not in diabetes mellitus between Hispanics and Non-Hispanic Blacks, with disparities in hypertension associated with socioeconomic status (family income, education, marital status) and also by alcohol, physical activity and age. However, race, education and BMI as class variables were statistically significantly associated with hypertension and diabetes mellitus p<0.0001. ^

Federally regulated institutional review boards: The aspects of a "model" IRB---Is the current system in need of a tune up or complete overhaul?

Institutional Review Boards (IRBs) are the primary gatekeepers for the protection of ethical standards of federally regulated research on human subjects in this country. This paper focuses on what general, broad measures that may be instituted or enhanced to exemplify a "model IRB". This is done by examining the current regulatory standards of federally regulated IRBs, not private or commercial boards, and how many of those standards have been found either inadequate or not generally understood or followed. The analysis includes suggestions on how to bring about changes in order to make the IRB process more efficient, less subject to litigation, and create standardized educational protocols for members. The paper also considers how to include better oversight for multi-center research, increased centralization of IRBs, utilization of Data Safety Monitoring Boards when necessary, payment for research protocol review, voluntary accreditation, and the institution of evaluation/quality assurance programs. ^ This is a policy study utilizing secondary analysis of publicly available data. Therefore, the research for this paper focuses on scholarly medical/legal journals, web information from the Department of Health and Human Services, Federal Drug Administration, and the Office of the Inspector General, Accreditation Programs, law review articles, and current regulations applicable to the relevant portions of the paper. ^ Two issues are found to be consistently cited by the literature as major concerns. One is a need for basic, standardized educational requirements across all IRBs and its members, and secondly, much stricter and more informed management of continuing research. There is no federally regulated formal education system currently in place for IRB members, except for certain NIH-based trials. Also, IRBs are not keeping up with research once a study has begun, and although regulated to do so, it does not appear to be a great priority. This is the area most in danger of increased litigation. Other issues such as voluntary accreditation and outcomes evaluation are slowing gaining steam as the processes are becoming more available and more sought after, such as JCAHO accrediting of hospitals. ^ Adopting the principles discussed in this paper should promote better use of a local IRBs time, money, and expertise for protecting the vulnerable population in their care. Without further improvements to the system, there is concern that private and commercial IRBs will attempt to create a monopoly on much of the clinical research in the future as they are not as heavily regulated and can therefore offer companies quicker and more convenient reviews. IRBs need to consider the advantages of charging for their unique and important services as a cost of doing business. More importantly, there must be a minimum standard of education for all IRB members in the area of the ethical standards of human research and a greater emphasis placed on the follow-up of ongoing research as this is the most critical time for study participants and may soon lead to the largest area for litigation. Additionally, there should be a centralized IRB for multi-site trials or a study website with important information affecting the trial in real time. There needs to be development of standards and metrics to assess the performance of the IRBs for quality assurance and outcome evaluations. The boards should not be content to run the business of human subjects' research without determining how well that function is actually being carried out. It is important that federally regulated IRBs provide excellence in human research and promote those values most important to the public at large.^

A qualitative assessment of multi-level nutrition strategies for reducing racial and ethnic disparities in diabetes

The purpose of this study is to examine the stages of program realization of the interventions that the Bronx Health REACH program initiated at various levels to improve nutrition as a means for reducing racial and ethnic disparities in diabetes. This study was based on secondary analyses of qualitative data collected through the Bronx Health REACH Nutrition Project, a project conducted under the auspices of the Institute on Urban Family Health, with support from the Centers for Disease Control and Prevention (CDC). Local human subjects' review and approval through the Institute on Urban Family Health was required and obtained in order to conduct the Bronx Health REACH Nutrition Project. ^ The study drew from two theoretical models—Glanz and colleagues' nutrition environments model and Shediac-Rizkallah and Bone's sustainability model. The specific study objectives were two-fold: (1) to categorize each nutrition activity to a specific dimension (i.e. consumer, organizational or community nutrition environment); and (2) to evaluate the stage at which the program has been realized (i.e. development, implementation or sustainability). ^ A case study approach was applied and a constant comparative method was used to analyze the data. Triangulation of data based was also conducted. Qualitative data from this study revealed the following principal findings: (1) communities of color are disproportionately experiencing numerous individual and environmental factors contributing to the disparities in diabetes; (2) multi-level strategies that targeted the individual, organizational and community nutrition environments can appropriately address these contributing factors; (3) the nutrition strategies greatly varied in their ability to appropriately meet criteria for the three program stages; and (4) those nutrition strategies most likely to succeed (a) conveyed consistent and culturally relevant messages, (b) had continued involvement from program staff and partners, (c) were able to adapt over time or setting, (d) had a program champion and a training component, (e) were integrated into partnering organizations, and (f) were perceived to be successful by program staff and partners in their efforts to create individual, organizational and community/policy change. As a result of the criteria-based assessment and qualitative findings, an ecological framework elaborating on Glanz and colleagues model was developed. The qualitative findings and the resulting ecological framework developed from this study will help public health professionals and community leaders to develop and implement sustainable multi-level nutrition strategies for addressing racial and ethnic disparities in diabetes. ^

An assessment of the relative influence of a vapor recovery system in reducing occupational exposure to volatile organic compounds in high performance liquid chromatography

Occupational exposures to organic solvents, specifically acetonitrile and methanol, have the potential to cause serious long-term health effects. In the laboratory, these solvents are used extensively in protocols involving the use of high performance liquid chromatography (HPLC). Operators of HPLC equipment may be potentially exposed to these organic solvents when local exhaust ventilation is not employed properly or is not available, which can be the case in many settings. The objective of this research was to characterize the various sites of vapor release in the HPLC process and then to determine the relative influence of a novel vapor recovery system on the overall exposure to laboratory personnel. The effectiveness of steps to reduce environmental solvent vapor concentrations was assessed by measuring exposure levels of acetonitrile and methanol before and after installation of the vapor recovery system. With respect to acetonitrile, the concentration was not statistically significant with p=0.938; moreover, exposure after the intervention was actually higher than prior to intervention. With respect to methanol, the concentration was not statistically significant with p=0.278. This indicates that the exposure to methanol after the intervention was not statistically significantly higher or lower than prior to intervention. Thus, installation of the vapor recovery device did not result in statistically significant reduction in exposures in the settings encountered, and acetonitrile actually increased significantly.^

Quantitative diffusion tensor imaging detects dopaminergic neuronal degeneration in a murine model of Parkinson's disease.

Early diagnosis of Parkinson's disease (PD) is required to improve therapeutic responses. Indeed, a clinical diagnosis of resting tremor, rigidity, movement and postural deficiencies usually reflect >50% loss of the nigrostriatal system in disease. In a step to address this, quantitative diffusion tensor magnetic resonance imaging (DTI) was used to assess nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication model of dopaminergic nigral degeneration. We now demonstrate increased average diffusion (p<0.005) and decreased fractional anisotropy (p<0.03) in the substantia nigra (SN) of 5- to 7-day MPTP-treated animals when compared to saline controls. Transverse diffusivity demonstrated the most significant differences (p < or = 0.002) and correlated with the numbers of SN dopaminergic neurons (r=-0.75, p=0.012). No differences were found in the striatum, corpus callosum, cerebral cortex, or ventricles. These results demonstrate that DTI may be used as a surrogate biomarker of nigral dopaminergic neuronal degeneration.