Associations of persistent organic pollutants in human adipose tissue with retinoid levels and their relevance to the redox microenvironment.

Humans are exposed to a myriad of chemical substances in both occupational and environmental settings. Persistent organic pollutants (POPs) have drawn attention for their adverse effects including cancer and endocrine disruption. Herein, the objectives were 1) to describe serum and adipose tissue retinol levels, along with serum retinol binding protein 4 (RBP4) concentrations, and 2) to assess the associations of adipose tissue POP levels with these retinoid parameters, as well as their potential interaction with the previously-observed POP-related disruption of redox microenvironment. Retinol was measured in both serum and adipose tissue along with RBP4 levels in serum samples of 236 participants of the GraMo adult cohort. Associations were explored by multivariable linear regression analyses and Weighted Quantile Sum regression. Polychlorinated biphenyls (PCBs) 180, 153 and 138 were related to decreased adipose tissue retinol levels and increased serum RBP4/retinol ratio. Dicofol concentrations > limit of detection were associated with decreased retinol levels in serum and adipose tissue. Additionally, increased adipose tissue retinol levels were linked to an attenuation in previously-reported associations of adipose tissue PCB-153 with in situ superoxide dismutase activity. Our results revealed a suggestive link between retinoids, PCBs and redox microenvironment, potentially relevant for both mechanistic and public health purposes.

In utero exposure to bisphenol-A disrupts key elements of retinoid system in male mice offspring.

The retinoid system controls essential cellular processes including mitosis, differentiation and metabolism among others. Although the retinoid-signalling pathway is a potential target for the action of several endocrine disrupting chemicals (EDCs), the information about the developmental effects of bisphenol-A (BPA) on the hepatic retinoid system is scarce. Herein, male mice were in utero exposed to BPA following maternal subcutaneous doses of 0, 10 and 100 µg/kg bw/day from gestational day 9-16 and they were sacrificed at post-natal day 30. Retinoid concentrations and gene expression of key elements involved in the retinoid system were determined in liver. BPA increased all-trans-retinoic acid concentration and expression of Adh1, Aox1 and Cyp1a2 (biosynthesis of retinoic acid), while reduced Mrp3 (efflux from hepatocyte to blood), increased Bcrp expression (biliary excretion) and changed the retinoid-dependent signalling system after reducing expression of Rxrß and increasing that of Fgf21. Furthermore, we found bivariate associations of Rarγ and Rxrγ expressions with all-trans-retinoic acid concentrations and of Fgf21 expression with that of Rarγ. Those findings occurred in animals which showed altered pancreatic function and impaired glucose metabolism during adulthood. The present information should be useful for enhancing testing methods for the identification of EDCs.

Comparative study on short- and long-term behavioral consequences of organophosphate exposure: relationship to AChE mRNA expression.

Organophosphates (OPs) affect behavior by inhibiting acetylcholinesterase (AChE). While the cognitive short-term effects may be directly attributed to this inhibition, the mechanisms that underlie OP's long-term cognitive effects remain controversial and poorly understood. Accordingly, two experiments were designed to assess the effects of OPs on cognition, and to ascertain whether both the short- and long-term effects of are AChE-dependent. A single subcutaneous dose of 250 mg/kg chlorpyrifos (CPF), 1.5mg/kg diisopropylphosphorofluoridate (DFP) or 15 mg/kg parathion (PTN) was administered to male Wistar rats. Spatial learning was evaluated 72 h or 23 weeks after exposure, and impulsive choice was tested at 10 and 30 weeks following OPs administration (experiment 1 and 2, respectively). Brain soluble and membrane-bound AChE activity, synaptic AChE-S mRNA, read-through AChE-R mRNA and brain acylpeptide hydrolase (APH) activity (as alternative non-cholinergic target) were analyzed upon completion of the behavioral testing (17 and 37 weeks after OPs exposure). Both short- and long-term CPF treatment caused statistically significant effects on spatial learning, while PTN treatment led only to statistically significant short-term effects. Neither CPF, DFP nor PTN affected the long-term impulsivity response. Long-term exposure to CPF and DFP significantly decreased AChE-S and AChE-R mRNA, while in the PTN treated group only AChE-S mRNA levels were decreased. However, after long-term OP exposure, soluble and membrane-bound AChE activity was indistinguishable from controls. Finally, no changes were noted in brain APH activity in response to OP treatment. Taken together, this study demonstrates long-term effects of OPs on AChE-S and AChE-R mRNA in the absence of changes in AChE soluble and membrane-bound activity. Thus, changes in AChE mRNA expression imply non-catalytic properties of the AChE enzyme.

Chronic dietary exposure to chlorpyrifos causes behavioral impairments, low activity of brain membrane-bound acetylcholinesterase, and increased brain acetylcholinesterase-R mRNA.

Chlorpyrifos (CPF) is an organophosphate (OP) insecticide that is metabolically activated to the highly toxic chlorpyrifos oxon. Dietary exposure is the main route of intoxication for non-occupational exposures. However, only limited behavioral effects of chronic dietary exposure have been investigated. Therefore, male Wistar rats were fed a dose of 5mg/kg/day of CPF for thirty-one weeks. Animals were evaluated in spatial learning and impulsivity tasks after 21 weeks of CPF dietary exposure and one week after exposure ended, respectively. In addition, the degree of inhibition of brain acetylcholinesterase (AChE) was evaluated for both the soluble and particulate forms of the enzyme, as well as AChE gene expression. Also, brain acylpeptide hydrolase (APH) was investigated as an alternative target for OP-mediated effects. All variables were evaluated at various time points in response to CPF diet and after exposure ended. Results from behavioral procedures suggest cognitive and emotional disorders. Moreover, low levels of activity representing membrane-bound oligomeric forms (tetramers) were also observed. In addition, increased brain AChE-R mRNA levels were detected after four weeks of CPF dietary exposure. However, no changes in levels of brain APH were observed among groups. In conclusion, our data point to a relationship between cognitive impairments and changes in AChE forms, specifically to a high inhibition of the particulate form and a modification of alternative splicing of mRNA during CPF dietary exposure.

Kinetics of inhibition of soluble peripheral nerve esterases by PMSF: a non-stable compound that potentiates the organophosphorus-induced delayed neurotoxicity.

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus carbamates and sulfonyl fluorides) is sometimes unable to yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work, kinetic data were obtained for phenylmethylsulfonyl fluoride (PMSF) tested at different concentrations incubated for up to 3 h with soluble fraction of chicken peripheral nerve. PMSF is a protease and esterase inhibitor causing protection or potentiation of the organophosphorus-induced delayed neuropathy and is unstable in water solution. The target of the promotion effect was proposed to be a soluble esterase not yet identified. A kinetic model equation was deduced assuming a multienzymatic system with three different molecular phenomena occurring simultaneously: (1) inhibition, (2) spontaneous chemical hydrolysis of the inhibitor and (3) ongoing inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied for analyzing the experimental data. The best-fitting model is compatible with a resistant component (16.5-18%) and two sensitive enzymatic entities (both 41%). The corresponding second-order rate constants of inhibition (ki = 12.04 × 10⁻² and 0.54 × 10⁻² µM⁻¹ min⁻¹, respectively) and the chemical hydrolysis constant of PMSF (kh = 0.0919 min⁻¹) were simultaneously estimated. These parameters were similar to those deduced in fixed-time inhibition experiments. The consistency of results in both experiments was considered an internal validation of the methodology. The results were also consistent with a significant ongoing inhibition. The proportion of enzymatic components showed in this work is similar to those previously observed in inhibition experiments with mipafox, S9B and paraoxon, demonstrating that this kinetic approach gives consistent results in complex enzymatic systems.

Inhibition with spontaneous reactivation of carboxyl esterases by organophosphorus compounds: paraoxon as a model.

In this work kinetic data were obtained for different paraoxon concentrations incubated with chicken serum and the soluble fraction of chicken peripheral nerve. A kinetic model equation was deduced by assuming a multienzymatic system with three different simultaneously occurring molecular phenomena: (1) inhibition; (2) simultaneous spontaneous reactivation; (3) "ongoing" inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied to analyze the experimental data versus the concentration of the inhibitor and the preincubation time in an inhibition experiment. The best-fitting model in the soluble fraction of chicken peripheral nerve was compatible with a resistant component (22%) and with two sensitive enzymatic entities (37 and 41%). The corresponding second-order rate constants of inhibition (k(i) = 1.8 × 10(-3) and 5.1 × 10(-3) nM(-1) min(-1), respectively) and the spontaneous reactivation constants (k(r) = 0.428 and 0.011 min(-1), respectively) were estimated. The best-fitting model in chicken serum was compatible with a resistant component (5.6%) and with two sensitive enzymatic entities (22.1 and 72.3%). The corresponding second-order rate constants of inhibition (k(i) = 5.8 × 10(-2) and 2.0 × 10(-3) nM(-1) min(-1), respectively) and the spontaneous reactivation constants (k(r) = 0.0044 and 0.0091 min(-1), respectively) were estimated. These parameters were similar to those observed in spontaneous reactivation experiments with preinhibited paraoxon samples. The consistency of the results of all the experiments is considered an internal validation of the methodology. The results are also consistent with a significant ongoing inhibition. The proportion of enzymatic components shown in this work by the inhibition and reactivation of paraoxon is similar to that previously observed in inhibition experiments with mipafox in both tissues, demonstrating that this kinetic approach provides consistent results in complex enzymatic systems. The high sensitivity (at nanomolar concentrations) of these esterases suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation.

Inhibition with spontaneous reactivation and the "ongoing inhibition" effect of esterases by biotinylated organophosphorus compounds: S9B as a model.

The biotinylated organophosphorus compound 1-(saligenin cyclic phospho)-9-biotinyldiaminononane (S9B) has been used for the detection, labeling and isolation of the membrane-bound neuropathy target esterase (NTE) as it was considered a specific inhibitor of NTE. After incubation with the soluble fraction of chicken peripheral nerve, most of the soluble esterase activity was highly sensitive to S9B, indicating NTE-like esterases. A kinetic model equation was used to assume a multi-enzymatic system with three different simultaneously occurring molecular phenomena; (1) inhibition; (2) simultaneous spontaneous reactivation; and (3) ongoing inhibition (inhibition during the substrate reaction); to fit the data to analyze kinetic behavior. A high "ongoing inhibition" effect was observed in an enzymatic component. A three-dimensional fit of the model was applied. The best fitting model is compatible with three sensitive enzymatic entities (33, 52 and 15%), and only one spontaneously reactivate. The second-order rate constants of inhibition (k(i)=116 x 10(6), 4.6 x 10(6) and 0.28 x 10(6)M(-1)min(-1), respectively) and the spontaneous reactivation constant for the first sensitive component (k(r)=0.0054 min(-1)) were simultaneously estimated. These parameters are similar to those deduced in spontaneous reactivation experiments of the preinhibited samples with S9B. The estimated proportions of enzymatic components are similar to those previously observed in inhibition experiments with mipafox, demonstrating that this kinetic approach offers consistent results.

Increase of R-/S-methadone enantiomer concentration ratio in serum of patients treated with either nevirapine or efavirenz.

An increased methadone enantiomer ratio (R/S) was associated to both nevirapine (179%, n=5) and efavirenz (36%, n=9) treatments when compared with that of controls (n=52). Additionally, in four follow-up patients, both R- and S-methadone normalized concentrations decreased (19%-93%) while R/S increased (22%-314%) following nevirapine/efavirenz treatment. R/S decreased (42%) after non-compliance with efavirenz treatment. Therefore, the methadone-maintenance-treatment outcome should be evaluated when patients are treated with drugs which are supposed to induce CYP3A4 and CYP2B6 isoforms.

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox. A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds.

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.

Detection of clinical interactions between methadone and anti-retroviral compounds using an enantioselective capillary electrophoresis for methadone analysis.

A capillary electrophoresis method was developed to detect interactions between methadone and anti-retroviral compounds. Eight subjects, who underwent methadone maintenance treatment in the Province of Alicante (Spain), consented to participate in the present study. Of those, one subject was followed up for 123 days to detect drug-drug interactions. The enantiomers of methadone and those of its main metabolite were conveniently resolved within 4 min using a chiral electrophoresis buffer mixture which consisted of phosphate buffer, pH 5, plus 0.2% highly sulphated-(beta)-cyclodextrin. The effective mobility of the analytes was in the 0.061-0.140 cm(2)/(kV s) range at pH 5. The R-methadone plasma concentration range for seven patients was 91-318 ng/mL, it decreased from 186 to 46 ng/mL in a patient followed-up on commencement of the anti-retroviral therapy, returning to the previous higher levels after progressive dose increases. We conclude that monitoring R-methadone plasma levels can be a useful tool for the dose adjustment of methadone.

[Prevalence of infection by HIV and hepatitis C virus in a cohort of patients on methadone treatment]. / Prevalencia de infección por virus de la inmunodeficiencia humana y hepatitis C en una cohorte de pacientes en tratamiento de mantenimiento con metadona.

BACKGROUND AND OBJECTIVES: Methadone maintenance treatment reduces drug-related-harm. The aims of the study were: a) to analyze the temporal tendencies of prevalence of infection by HIV and hepatitis C virus, and b) to evaluate if the adherence to methadone maintenance treatment protects against the infection by HIV and hepatitis C virus. PATIENTS AND METHOD: Retrospective longitudinal study with All the 1,487 patients receiving treatment in the Province of Alicante between June 1990 and December 1997. The prevalence of infection by HIV and hepatitis C virus were computed. The variables associated to the infection by HIV and hepatitis C virus were recognized though a model of logistic regression. RESULTS: The prevalence of infection by HIV decreased, and the prevalence of infection by hepatitis C virus increased along the studied period. The independent predictors with regard to the infection by HIV were the age of beginning in treatment (odds ratio [OR] = 0.95; 95% confident interval [95%CI], 0.93-0.97; p < 0.001), total time within treatment (OR = 0.99; 95%CI, 0.98-1.0; p = 0.01) and non-parenteral route of administration (OR = 0.25; 95%CI, 0.18-0.33; p < 0.001). CONCLUSIONS: To be admitted to methadone maintenance treatment for the first time after opiate dependence has been developed and to remain in treatment for long periods of time, all that decreases the risk of infection by HIV.

Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of serum esterases are very similar to those of soluble fraction of peripheral nerves. So, serum PVases could be considered as appropriate biomarkers, as a mirror for the neural soluble paraoxon and mipafox sensitive soluble esterases that could be used for biomonitoring purpose.

Survival study of opioid addicts in relation to its adherence to methadone maintenance treatment.

OBJECTIVES: To study the putative role of methadone maintenance treatment in the improvement of life expectancy of opioid addicts. DESIGN: Retrospective longitudinal study. PARTICIPANTS: All 1487 patients receiving methadone maintenance treatment in Alicante between June 1990 and December 1997. STATISTICAL ANALYSIS: Mortality rates were studied using Kaplan-Meier survival curves. Protection or risk factors were analyzed using Cox's proportional hazards model. RESULTS: Mortality rates decreased from 87/1000 in 1991 to 17/1000 in 1997. The following factors influenced mortality: HIV infection [Hazard Ratio (HR)=7, 95% confidence interval (CI)=4-12]; current methadone status (HR=3.2, 95%CI=1.5-7.1) and MMT retention (retained vs. drop-out, HR=0.5, 95%CI=0.2-1.1; re-enrolled vs. drop-out, HR=0.3, 95%CI=0.2-0.5). CONCLUSION: Expediting entry and re-enrolling in methadone maintenance treatment improves survival.

Prevalencia de infección por virus de la inmunodeficiencia humana y hepatitis Cen una cohorte de pacientes en tratamiento de mantenimiento con metadona / Prevalence of infection by HIV and hepatitis C virus in a cohort of patients on methadone treatment

FUNDAMENTO Y OBJETIVOS: El tratamiento de mantenimiento con metadona reduce los daños asociados al consumo de drogas. Los objetivos de este trabajo han sido: a) analizar la tendencia temporal de la infección por el virus de la inmunodeficiencia humana (VIH) y de la hepatitis C, y b) valorar si permanecer vinculado al tratamiento de mantenimiento con metadona protege la infección por el VIH y la hepatitis C. PACIENTES Y MÉTODO: Estudio longitudinal retrospectivo con 1.487 pacientes de la provincia de Alicante que recibieron tratamiento de mantenimiento con metadona entre 1990 y 1997. Se calculó la frecuencia de infección por VIH y hepatitis C. Las variables asociadas a la infección por el VIH y la hepatitis C se identificaron mediante un modelo de regresión logística. RESULTADOS: La prevalencia de infección por el VIH disminuyó, mientras que la prevalencia de infección por hepatitis C aumentó durante el período estudiado. Los predictores independientes asociados a la infección por el VIH fueron la edad de inicio en tratamiento (odds ratio [OR] = 0,95; intervalo de confianza [IC] del 95 por ciento, 0,93-0,97; p < 0,001), el tiempo total en tratamiento (OR = 0,99; IC del 95 por ciento, 0,98-1,0; p = 0,01) y la vía de administración no parenteral (OR = 0,25; IC del 95 por ciento, 0,18-0,33; p < 0,001). CONCLUSIONES: En pacientes con dependencia de opiáceos que reciben tratamiento con metadona se observa que el riesgo de infección por el VIH se reduce en los sujetos que iniciaron el tratamiento en una edad temprana y en aquellos con períodos largos de tratamiento (AU)

Estudio de costes de un tratamiento de mantenimiento con metadona de bajo nivel de prestaciones / Cost study of a methadone maintenance treatment

Fundamento: Los tratamientos con metadona a individuos drogodependientes tienen un coste poco conocido. Pacientes y métodos: El coste se calculó aplicando el sistema de información económica de la Conselleria de Sanitat (1997). Resultados: El coste de la dosis de metadona fue de 76 ptas., el de la primera visita de 4.517 ptas., las determinaciones de orina de 760 ptas. y el desplazamiento de la unidad móvil costó 18.895 ptas. Conclusiones: El sistema de cálculo es aplicable al tratamiento de mantenimiento con metadona a individuos drogodependientes. El coste es bajo. (AU)