Pain management in internal medicine and effects of a standardised educational intervention: the FADOI-DOMINO study.

PURPOSE: Few real-world data are available on the frequency and management of pain in Internal Medicine (IM). Aims of our study were to assess the prevalence of pain in IM, and to evaluate the effects on pain management of a standardised educational programme. MATERIALS AND METHODS: The study was performed in 26 IM Units in Italy, with two cross-sectional surveys (PRE phase and POST phase) interspersed with an educational programme. In PRE phase each Centre reviewed the hospital charts of the last 100 consecutive patients hospitalised for any cause. An educational programme was conducted in each Centre by means of the 'outreach visit', a face-to-face meeting between health personnel and a trained external expert. Six months after, each Centre repeated the data collection (POST phase), specular to the PRE. RESULTS: A total of 5200 medical charts were analysed. Pain was documented in 37.5% of the patients. After the educational intervention, the intensity of pain was appropriately assessed in a higher percentage of patients (77.4% vs. 47.8%, p = 0.0001), and it was more frequently monitored during hospitalisation. Qualitative definition of pain (pathogenesis, duration, etc.) increased in POST phase (75.4% vs. 62.7%, p = 0.0001). A 73.3% increase in the use of strong opioids was detected following educational programme. CONCLUSIONS: Pain affects 4 out of 10 patients hospitalised in IM. According to our large real-world study, to implement a standardised one-shot educational programme may persistently improve the attitude of health personnel towards the characterisation and management of pain.

Opioid-related bowel dysfunction: prevalence and identification of predictive factors in a large sample of Italian patients on chronic treatment.

BACKGROUND AND OBJECTIVES: Opioid-bowel dysfunction (OBD) is a broad range of symptoms potentially associated with opioid therapy. This prospective, multicentric study assesses the prevalence of OBD in patients on analgesic therapy for the treatment of pain from any cause and identifies the factors associated with the onset of this side effect. MATERIALS AND METHODS: Consecutive patients aged > 18 years, on analgesic treatment with opioids, non-steroidal anti-inflammatory drugs (NSAIDs) or other therapies for chronic pain of any aetiology were included in the study. The association of OBD with gender, age, pain aetiology and analgesic treatment was analyzed by multivariate analysis and logistic analysis. RESULTS: In total, 2324 patients were included in the study. The prevalence of OBD symptoms was 63.5%, despite that the wide majority of patients (89.5%) were receiving laxatives. OBD symptoms were judged as directly correlated with analgesic therapy in 85.1% of cases. The highest prevalence of constipation was reported with morphine, whereas the lowest was observed in patients on oxycodone CR and buprenorphine TTS. Statistical analysis showed that patients on opioids have a higher likelihood of experiencing OBD symptoms than those on NSAIDs or other treatments (66.2% vs 37.0%), and this probability is even higher in those with cancer-related pain (69.3%). Female gender and age > 70 years also appeared as risk factors. The logistic analysis indicated that cancer-related pain, increased age and the use of fentanyl are positive predictors of the presence of OBD, whereas the administration of oxycodone CR was associated with a decreased incidence of these symptoms. DISCUSSION: Even with the limitation of any observational experience, this study suggests, for the first time, the existence of some factors predictive of the onset of OBD symptoms in patients on analgesic treatment. Moreover, different opioids seem to be associated with a different risk of experiencing these symptoms.

The management of constipation in palliative care: clinical practice recommendations.

Constipation is one of the most common problems in patients receiving palliative care and can cause extreme suffering and discomfort. The aims of this study are to raise awareness of constipation in palliative care, provide clear, practical guidance on management and encourage further research in the area. A pan-European working group of physicians and nurses with significant experience in the management of constipation in palliative care met to evaluate the published evidence and produce these clinical practice recommendations. Four potentially relevant publications were identified, highlighting a lack of clear, practical guidance on the assessment, diagnosis and management of constipation in palliative care patients. Given the limited data available, our recommendations are based on expert clinical opinion, relevant research findings from other settings and best practice from the countries represented. Palliative care patients are at a high risk of constipation, and while general principles of prevention should be followed, pharmacological treatment is often necessary. The combination of a softener and stimulant laxative is generally recommended, and the choice of laxatives should be made on an individual basis. The current evidence base is poor and further research is required on many aspects of the assessment, diagnosis and management of constipation in palliative care.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Absorption of fumonisin B1 and aminopentol on an in vitro model of intestinal epithelium; the role of P-glycoprotein.

The aim of the present paper is to evaluate the absorption of fumonisin B1 and its principal metabolite, aminopentol on a human intestinal model, Caco-2 cells, cultured on semi-permeable inserts, that reproduces the two different intestinal compartments: luminal (apical) and serosal (basolateral) side. Following separate exposure in apical and in basolateral compartments, aminopentol passage through the cell layer (in particular from basolateral to apical direction) was shown, while it was not observed for the parent compound. The different aminopentol distribution between the two compartments of the culture system, and its variation in presence of verapamil or probenecid (P-gp and MRP inhibitors respectively), strongly suggests the involvement of P-glycoprotein in the influx/efflux mechanisms of aminopentol in the intestinal cells, reducing its oral bioavailability.

Evaluation of Fumonisin B(1) and its metabolites absorption and toxicity on intestinal cells line Caco-2.

The aim of the present paper is to investigate intestinal absorption and toxicity of Fumonisin B(1) (FB(1)) and its partially (PHFB(1) and PHFB(2)) and totally hydrolyzed (HFB(1)) metabolites, using the human intestinal cell line Caco-2, a very well known in vitro model of intestinal epithelium for absorption and metabolism studies. Caco-2 cells were treated for 48 h with several toxin concentrations (in the range of 1-138 microM). At the end of exposure period, no significant variation on cell viability has been observed with all chemicals tested, either in undifferentiated cells or in differentiated ones, suggesting a poor toxicity of these mycotoxins for intestinal cells. In any case, FB(1) appears the most active in this respect. For which concerns the cellular absorption, FB(1), PHFB(1) and PHFB(2) are never detected into Caco-2 cells. On the contrary, a dose-dependent absorption of HFB(1) has been observed in differentiated cells, which express enzymatic and metabolic characteristics of mature enterocytes. Thus HFB(1), losing the tricarballylic acid chain, is more bioavailable than FB(1) on intestinal cell, supporting the hypothesis that in risk evaluation of fumonisins exposure its metabolites are also relevant.

Toxicology investigations with cell culture systems.

This review concerns some of the cell culture systems that are most frequently used in toxicology investigations. In particular, it sets out to evaluate the effectiveness of these cell culture systems in assessing the toxic potential of chemicals. Metabolic studies and general and specific toxicology investigations are highlighted. Specific toxicology investigations relate to the effects of the tests substances on the highly specialized functions typical of the cell systems chosen. The general toxicology investigations include most of the other studies where differentiated or undifferentiated cells have been used to evaluate the effects of the tested substances on common basic biochemical processes essential for life. Lastly, we have attempted to focus attention on the most promising applications of cell cultures in toxicology studies for the near future and to identify those areas where further research is needed. Because of the several excellent reviews that already exist, we have decided not to consider cell cultures utilized in screening potential mutagens and carcinogens. We have also excluded investigations of drug therapeutic effects and action mechanisms of drugs.

Cytotoxic effects of wheat gliadin-derived peptides.

The peptic-tryptic-cotazym (PTC) digest, obtained from bread wheat gliadin by simulating in vivo protein digestion, was more active than the PTC-digest of durum wheat gliadin in reversibly inhibiting HEp-2 cell proliferation and in increasing cellular acid phosphatase. Colony-forming ability of the cells was not affected by treatment with both bread or durum wheat gliadin peptides. The peptic-tryptic (PT) digest of bread wheat gliadin also showed agglutinating activity of HEp-2 cells.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

In vitro toxicology methods: impact on regulation from technical and scientific advancements.

The impressive advancement of technologies in biomedical research, and particularly in the area of in vitro experimental models, has opened up new possibilities related to co-cultures, micromass or stem cell cultures. Engineered cells to study specific targets and/or mechanisms are also available. Moreover, a very subtle approach in the study of toxicological effects is represented by the very recent genomics and proteomics techniques. New mechanistically based methods could be established from all these approaches, which, once validated, could enter the regulatory procedure. So far, in toxicology, only a few in vitro tests are accepted for regulatory purposes, such as those related to corrosion, phototoxicity and absorption. Many others are in the pre-validation or validation phase. An area where in vitro tests play a key role is the genetic toxicology. In this context, the most recent testing strategies and test methods will be presented, with particular attention to the recently updated guidelines for food additives by the EU Scientific Committee on Food. An improvement in the implementation of validated methods could arise from a better coordination on the matter at national and international levels, the harmonisation of different legislations, and a strict control of the national rules in order to make them up-to-date with respect to validated methods.

Effects of gliadin-derived peptides from bread and durum wheats on in vitro cultures of human cell lines. Implications for coeliac disease pathogenesis.

A peptic-tryptic-cotazym digest, obtained from bread (hexaploid) wheat gliadins under experimental conditions mimicking in vivo protein digestion, was found to reduce in vitro viability of human embryo (MRC-5) and tumor cell (Hep-2) lines. Time of onset and extent of cytotoxic effects were largely dependent on initial peptide concentrations in the culture medium. The presence of 2% fetal calf serum was capable of delaying, but not of preventing, the onset of cytotoxic effects only in MRC-5 cultures. A peptic-tryptic-cotazim digest obtained from durum (tetraploid) wheat gliadins and tested under identical conditions did not show any cytotoxic activity on MRC-5 and Hep-2 cell lines. These results indicate that cell systems are useful to investigate pathogenetic mechanisms of coeliac disease (gluten-dependent enteropathy).

Freshly isolated cells and cell lines from the intestine as an in vitro model for toxicological studies.

The intestine is a crucial organ in respect of toxicity of ingested compounds, because of its extensive exposed surface area as well as its absorptive and physiological properties. Only recently has attention been paid to this organ in the investigation of pathological conditions using in vitro models. The first studies performed dealt with the metabolic capacity of isolated intestinal cells and date back to 1977. More recently, intestinal cell lines have been characterized for the presence of some activating-deactivating enzymes. Cytotoxicity studies reported in the literature predominantly concern screening of antitumoral drugs using carcinoma cell lines and investigation of the mechanisms involved. A variety of other chemicals with possible toxic effects have been studied in intestinal cells and these include food contaminants, anti-infective drugs, natural toxins and dietary compounds. However those studies are still very limited in number and represent a wide range of approaches, in spite of the fact that very interesting models of intestinal cells are now available and the technology is improving.

Functional alterations induced by the food contaminant furazolidone on the human tumoral intestinal cell line Caco-2.

Caco-2 cells, which are derived from a human colon carcinoma and are able to differentiate in culture, have been used to study the effect of furazolidone (FZ), a chemical belonging to the nitrofuran family which is frequently used for the prevention of animal infections. Its potentially toxic residues could remain in some food products of animal origin and affect human health. Toxicity has been measured by different parameters, either in undifferentiated cells (day 7 of culture), or on differentiated cells (day 21 of culture). Our results indicate that FZ may seriously affect the proliferating portion of the intestinal mucosa, while the differentiated cells appear to be more resistant. However, the slight effect recorded on the aspecific and specific functions of the differentiated cells may suggest that the specialized portion of the intestine can also be compromised by the drug. Caco 2 cells seem a good model for a deeper investigation of the mechanism involved in the toxic action of FZ.

Toxicology investigations with cell culture systems: 20 years after.

From almost 20 years the "in vitro" model has gained a wide ground in toxicological investigation, providing advanced tools, reliable protocols, mechanistic information. These advancements have been done thanks to different approaches, addressed at improving chemical testing and validating procedures, at exploring the cellular and molecular basis of toxicity, at studying the modifications that xenobiotics undergo in the cellular environment. In this review the most advanced cellular models, the mechanisms of cell death, the techniques to monitor gene activation, following chemical exposure, is highlighted. Moreover the more recent in vitro models to approach the biotransformation issue will be presented.

Cytotoxicity of halogenated benzenes and its relationship with logP.

The in vitro toxicity of a series of environmentally relevant halobenzenes was tested using a Chinese hamster lung fibroblast cell line and its relationship with the logarithm of octanol/water partition coefficient (logP) was investigated. Since we wanted to study the direct biological activity of the parent substances, we have used the V-79 cell line that does not express phase I metabolic activities. Moreover, because of the available knowledge on the substances, we decided to perform the colony-forming ability test (CFA) and to analyse the DNA damage by a cytofluorimetric assay. To identify the concentration range at which the toxic effect could be detected, a prescreen with the neutral red assay has been performed. All the substances tested were positive in the CFA, but, according to the concentration values inhibiting this activity by 50%, they can be divided into two groups of differing toxicity. The FACScan analysis shows for the majority of the halobenzenes a clear hypodiploid peak. A good correlation between values describing the concentration that inhibits CFA by 50% and logP was found, indicating that it depends on the hydrophobic properties of the compounds and that logP is a suitable descriptor.

Choice and standardization of test protocols in cytotoxicology: A multicentre approach.

A major problem that interferes with the introduction of in vitro tests for toxicological risk assessment is that of defining reliable standardized protocols. This issue was approached in the present study with an interlaboratory comparison of three cytotoxicity assays detecting chemical toxicity as impairment of cell viability in confluent cultures, reduction of colony forming ability, and inhibition of cell proliferation over 3 days of treatment. The study was performed using V79 cells, which are unable to activate indirectly-acting xenobiotics, and six chemicals with different mechanisms of action: two antioxidants (butylated hydroxyanisole and butylated hydroxytoluene), an inhibitor of protein synthesis (cycloheximide), an alkylating agent requiring metabolic activation (cyclophosphamide), an uncoupler of oxidative phosphorylation (dinitrophenol), and a genotoxic metal salt (potassium dichromate). The three tests produced the same rank of relative toxic potency for the tested chemicals, based on LC(50) values. The cell viability test appeared to be the most suited for the screening of unknown chemicals, given its simplicity and better reproducibility.

Investigation of the cytotoxicity produced by generation of short-lived reactive metabolites in vitro: A study with paracetamol.

A V79 cell incubation, incorporating rat or hamster liver 9000 g supernatant (S-9) or microsomal fraction and used previously to detect the toxicity of reactive metabolites of cyclophosphamide and bromobenzene, has been used to examine the toxicity of short-lived reactive metabolites of paracetamol. Cytotoxicity was observed in the absence of an activating system and did not increase when an activating system was included in the incubation, even when this was derived from the livers of hamsters treated with beta-naphthoflavone (to increase the activity of the cytochrome P-450 form responsible for paracetamol activation) and diethyl maleate (to deplete protective glutathione stores). The failure to detect metabolism-mediated cytotoxicity of paracetamol in this assay system may be related to the high reactivity of the toxic metabolite. This, in turn, suggests that other systems, based on activation within target cells or on the detection of an endpoint closer to the activation event, are required for the in vitro detection of short-lived cytotoxic metabolites.

Nitric oxide production in Caco-2 cells exposed to different inducers, inhibitors and natural toxins.

The involvement of the NO pathway in several intestinal inflammatory diseases is under investigation. In vitro models may provide a useful approach to better characterise this pathway at the cellular level. For this purpose, we have used Caco-2 cells, which are able to spontaneously differentiate in long-term culture to small intestine enterocytes. The effect of different NO pathway inducers [gamma-interferon (IFN-gamma) and phorbol myristate acetate (PMA)] has been studied. Our results demonstrate that Caco-2 cells constitutively express NOS at very low levels, while the induction with PMA+IFN-gamma triggers the expression of the inducible isoform with a stronger effect starting from day 14 of differentiation. The use of specific inhibitors of gene expression, at transcriptional and translational level, suggests that new synthesis of iNOS mRNA is required, through direct activation of the gene or new synthesis of transcription-required factors, as indicated by CHX inhibition. The morphological alteration induced by PMA+IFN-gamma is reversed by iNOS inhibitor, suggesting that the NO pathway may be involved in the cytoskeletal alterations. The DSP toxins, OA and DTX-1, induce NO production at levels corresponding to their different toxicity, previously detected in Caco-2 cells.

Evaluation of metabolic endpoints of acute cytotoxicity in V79 fibroblasts.

A group of cytotoxicity tests that detect alterations in cell metabolism were applied to obtain a preliminary classification of test chemicals, based on their main mechanism of toxicity. V79 cells were exposed to toxic compounds in 'acute' treatments (up to 2 hr) and the specificity of different endpoints of cytotoxicity was compared. We tested five directly acting chemicals: butylated hydroxyanisole, butylated hydroxytoluene, cycloheximide, potassium dichromate [Cr(VI)] and dinitrophenol using four assays measuring; [(3)H]thymidine uptake and incorporation into DNA, [(3)H]leucine incorporation into proteins, ATP pool size and cellular energy charge, and oxygen consumption. In the thymidine-uptake test the radioactivity of the nucleotide pool was hardly affected by low concentrations of the toxic chemicals and was reduced by about 20-30% at the 10(-4)m concentration, except by butylated hydroxytoluene; the latter which caused a marked inhibition of [(3)H]thymidine uptake. Thymidine incorporation into DNA was more specifically altered, showing a net inhibition by potassium dichromate, in a concentration-dependent fashion, and by butylated hydroxytoluene. Protein synthesis was more inhibited by potassium dichromate and butylated hydroxytoluene than by the specific inhibitor, cycloheximide. All chemicals reduced ATP concentration, but the energy charge was scarcely affected, reflecting a parallel decrease of the other adenine nucleotides. The assay measuring early changes in oxygen consumption produced the expected results with dinitrophenol, the antioxidants and potassium dichromate, and showed that cycloheximide also inhibits mitochondrial respiration, in agreement with the observed fall of intracellular ATP. All the chemicals tested induced a range of effects on the metabolic parameters analysed but specific pathways of toxicity may be inferred by comparing the results of the individual tests.

Established cell lines for safety assessment of food contaminants: differing furazolidone toxicity to V 79, HEp-2 and Caco-2 cells.

In vitro models, preferentially derived from human tissues, may be valuable tools to study the biotransformation and toxicity of compounds that may be present as residues in food products. Such residues may represent a risk to human health, and therefore call for increased testing. Three established cell lines were used to study the toxic effect of furazolidone (FZ), a widely used veterinary drug: HEp-2 cells, derived from a human larynx carcinoma, previously used in toxicity screening of several compounds; Caco-2 cells, derived from a human colon adenocarcinoma, able to differentiate partially in culture, and V 79, a fibroblast cell line derived from Chinese hamster lung, widely used to assess direct toxicants. Various toxicity parameters were used, primarily dealing with cell death and cell proliferation. In all cell lines FZ at a concentration of 5 micrograms/ml caused a marked decrease in cell viability and especially in cell proliferation. Inhibition of DNA synthesis has also been observed, even if at higher concentrations. However, only in V 79 cells was the decrease in cell number accompanied by a marked increase in lactate dehydrogenase leakage due to membrane damage. Moreover, the surviving V 79 cells, after removal of FZ, fully recovered from the effect of the drug, as shown by their full capacity to attach to dishes and to form colonies. Surviving cells of the other two cell lines showed much poorer colony-forming ability. Exposure of Caco-2 cells and, to a lesser extent, HEp-2 cells, caused a marked increase in oxygen consumption, that possibly was due to redox cycling of the initially formed radical nitro anion. Biotransformation of the drug by all three cell lines was accompanied by the formation of protein-bound metabolites, HEp-2 being the most active cells. The toxic effects recorded show that cell lines provide a sensitive system in toxicity assessment. Moreover, it may be suggested that a battery of cell lines, including some of human origin, as well as a battery of endpoints, may be of help in addressing further specific mechanistic investigations.