Treating colorectal peritoneal metastases with an injectable cytostatic loaded supramolecular hydrogel in a rodent animal model.

Patients with peritoneal metastases (PM) of colorectal cancer have a very poor outcome. Intraperitoneal delivery of chemotherapy is the preferred route for PM treatment. The main limitation of the treatment options is the short residence time of the cytostatic, with subsequent short exposure of the cancer cells. To address this, a supramolecular hydrogel has been developed that allows both local and slow release of its encapsulated drug, mitomycin C (MMC) or cholesterol-conjugated MMC (cMMC), respectively. This experimental study investigates if drug delivery using this hydrogel improves the therapeutic efficacy against PM. PM was induced in WAG/Rij rats (n = 72) by intraperitoneally injecting syngeneic colon carcinoma cells (CC531) expressing luciferase. After seven days, animals received a single intraperitoneal injection with saline (n = 8), unloaded hydrogel (n = 12), free MMC (n = 13), free cMMC (n = 13), MMC-loaded hydrogel (n = 13), or cMMC-loaded hydrogel (n = 13). Primary outcome was overall survival with a maximum follow-up of 120 days. Intraperitoneal tumor development was non-invasive monitored via bioluminescence imaging. Sixty-one rats successfully underwent all study procedures and were included to assess therapeutic efficacy. After 120 days, the overall survival in the MMC-loaded hydrogel and free MMC group was 78% and 38%, respectively. A trend toward significance was found when comparing the survival curves of the MMC-loaded hydrogel and free MMC (p = 0.087). No survival benefit was found for the cMMC-loaded hydrogel compared to free cMMC. Treating PM with our MMC-loaded hydrogel, exhibiting prolonged MMC exposure, seems effective in improving survival compared to treatment with free MMC.

Fluxomics reveals cellular and molecular basis of increased renal ammoniagenesis.

The kidney plays a critical role in excreting ammonia during metabolic acidosis and liver failure. The mechanisms behind this process have been poorly explored. The present study combines results of in vivo experiments of increased total ammoniagenesis with systems biology modeling, in which eight rats were fed an amino acid-rich diet (HD group) and eight a normal chow diet (AL group). We developed a method based on elementary mode analysis to study changes in amino acid flux occurring across the kidney in increased ammoniagenesis. Elementary modes represent minimal feasible metabolic paths in steady state. The model was used to predict amino acid fluxes in healthy and pre-hyperammonemic conditions, which were compared to experimental fluxes in rats. First, we found that total renal ammoniagenesis increased from 264 ± 68 to 612 ± 87 nmol (100 g body weight)-1 min-1 in the HD group (P = 0.021) and a concomitated upregulation of NKCC2 ammonia and other transporters in the kidney. In the kidney metabolic model, the best predictions were obtained with ammonia transport as an objective. Other objectives resulting in a fair correlation with the measured fluxes (correlation coefficient >0.5) were growth, protein uptake, urea excretion, and lysine and phenylalanine transport. These predictions were improved when specific gene expression data were considered in HD conditions, suggesting a role for the mitochondrial glycine pathway. Further studies are needed to determine if regulation through the mitochondrial glycine pathway and ammonia transporters can be modulated and how to use the kidney as a therapeutic target in hyperammonemia.

Chemosensitivity of 3D Pancreatic Cancer Organoids Is Not Affected by Transformation to 2D Culture or Switch to Physiological Culture Medium.

Organoids are increasingly used to investigate patient-specific drug responsiveness, but organoid culture is complex and expensive, and carried out in rich, non-physiological media. We investigated reproducibility of drug-responsiveness of primary cell cultures in 2D versus 3D and in conventional versus physiological cell culture medium. 3D pancreatic ductal adenocarcinoma organoid cultures PANCO09b and PANCO11b were converted to primary cell cultures growing in 2D. Transformed 2D cultures were grown in physiological Plasmax medium or Advanced-DMEM/F12. Sensitivity towards gemcitabine, paclitaxel, SN-38, 5-fluorouacil, and oxaliplatin was investigated by cell viability assays. Growth rates of corresponding 2D and 3D cultures were comparable. PANCO09b had a shorter doubling time in physiological media. Chemosensitivity of PANCO09b and PANCO11b grown in 2D or 3D was similar, except for SN-38, to which PANCO11b cultured in 3D was more sensitive (2D: 8.2 ×10-3 ± 2.3 ×10-3 vs. 3D: 1.1 ×10-3 ± 0.6 ×10-3, p = 0.027). PANCO09b and PANCO11b showed no major differences in chemosensitivity when cultured in physiological compared to conventional media, although PANCO11b was more sensitive to SN-38 in physiological media (9.8 × 10-3 ± 0.7 × 10-3 vs. 5.2 × 10-3 ± 1.8 × 10-3, p = 0.015). Collectively, these data indicate that the chemosensitivity of organoids is not affected by culture medium composition or culture dimensions. This implies that organoid-based drug screens can be simplified to become more cost-effective.

Temporal Transcript Profiling Identifies a Role for Unfolded Protein Stress in Human Gut Ischemia-Reperfusion Injury.

BACKGROUND & AIMS: Intestinal ischemia-reperfusion injury is a serious and life-threatening condition. A better understanding of molecular mechanisms related to intestinal ischemia-reperfusion injury in human beings is imperative to find therapeutic targets and improve patient outcome. METHODS: First, the in vivo dynamic modulation of mucosal gene expression of the ischemia-reperfusion-injured human small intestine was studied. Based on functional enrichment analysis of the changing transcriptome, one of the predominantly regulated pathways was selected for further investigation in an in vitro human intestinal organoid model. RESULTS: Ischemia-reperfusion massively changed the transcriptional landscape of the human small intestine. Functional enrichment analysis based on gene ontology and pathways pointed to the response to unfolded protein as a predominantly regulated process. In addition, regulatory network analysis identified hypoxia-inducing factor 1A as one of the key mediators of ischemia-reperfusion-induced changes, including the unfolded protein response (UPR). Differential expression of genes involved in the UPR was confirmed using quantitative polymerase chain reaction analysis. Electron microscopy showed signs of endoplasmic reticulum stress. Collectively, these findings point to a critical role for unfolded protein stress in intestinal ischemia-reperfusion injury in human beings. In a human intestinal organoid model exposed to hypoxia-reoxygenation, attenuation of UPR activation with integrated stress response inhibitor strongly reduced pro-apoptotic activating transcription factor 4 (ATF4)-CCAAT/enhancer-binding protein homologous protein (CHOP) signaling. CONCLUSIONS: Transcriptome analysis showed a crucial role for unfolded protein stress in the response to ischemia-reperfusion in human small intestine. UPR inhibition during hypoxia-reoxygenation in an intestinal organoid model suggests that downstream protein kinase R-like ER kinase (PERK) signaling may be a promising target to reduce intestinal ischemia-reperfusion injury. Microarray data are available in GEO (https://www.ncbi.nlm.nih.gov/gds, accession number GSE37013).

A novel human cell culture model to study visceral smooth muscle phenotypic modulation in health and disease.

Adaptation of the smooth muscle cell (SMC) phenotype is essential for homeostasis and is often involved in pathologies of visceral organs (e.g., uterus, bladder, gastrointestinal tract). In vitro studies of the behavior of visceral SMCs under (patho)-physiological conditions are hampered by a spontaneous, uncontrolled phenotypic modulation of visceral SMCs under regular tissue culture conditions. We aimed to develop a new visceral SMC culture model that allows controlled phenotypic modulation. Human uterine SMCs [ULTR and telomerase-immortalized human myometrial cells (hTERT-HM)] were grown to confluency and kept for up to 6 days on regular tissue culture surfaces or basement membrane (BM) matrix-coated surfaces in the presence of 0-10% serum. mRNA and protein expression and localization of SMC-specific phenotype markers and their transcriptional regulators were investigated by quantitative PCR, Western blotting, and immunofluorescence. Maintaining visceral SMCs confluent for 6 days increased α-smooth muscle actin (1.9-fold) and smooth muscle protein 22-α (3.1-fold), whereas smooth muscle myosin heavy chain was only slightly upregulated (1.3-fold). Culturing on a BM matrix-coated surface further increased these proteins and also markedly promoted mRNA expression of γ-smooth muscle actin (15.0-fold), smoothelin (3.5-fold), h-caldesmon (5.2-fold), serum response factor (7.6-fold), and myocardin (8.1-fold). Whereas additional serum deprivation only minimally affected contractile markers, platelet-derived growth factor-BB and transforming growth factor ß1 consistently reduced versus increased their expression. In conclusion, we present a simple and reproducible visceral SMC culture system that allows controlled phenotypic modulation toward both the synthetic and the contractile phenotype. This may greatly facilitate the identification of factors that drive visceral SMC phenotypic changes in health and disease.

Cyclooxygenase-2 Is Essential for Colorectal Anastomotic Healing.

OBJECTIVE: To study the effects of COX-2 on colonic surgical wound healing. BACKGROUND: Cyclooxygenase-2 (COX-2) is a key enzyme in gastrointestinal homeostasis. COX-2 inhibitors have been associated with colonic anastomotic leakage. METHODS: Wildtype, COX-2 knockout and COX-2 heterozygous mice were subjected to a model of colonic anastomotic leakage, and were treated with vehicle, diclofenac, or prostaglandin E2 (PGE2), the most important COX-2 product in the intestine. We assessed anastomotic leakage, mortality, angiogenesis, and inflammation. Furthermore, we investigated the association between anastomotic leakage and a human polymorphism of the COX-2 gene resulting in low COX-2 levels. RESULTS: Diclofenac, a nonsteroidal anti-inflammatory drug inhibiting COX-2, increased anastomotic leakage compared to vehicle-treated mice (100% vs 25%, respectively). Similarly, 92% of COX-2-deficient mice developed anastomotic leakage (P = 0.003) compared to WT. PGE2 partly rescued this severe phenotype because only 46% of PGE2-administered COX-2 knockout mice developed anastomotic leakage (P = 0.02). This may be related to decreased neovascularization, because decreased CD31 staining, indicating less blood vessels, was observed in COX-2 mice (2 vessels/mm vs 6 vessels/mm in controls (P = 0.03)). This effect could partly be reversed by administration of PGE2 to COX-2 mice. No significant differences in inflammation were found. PTGS2-765G>C polymorphism in humans, associated with reduced COX-2 expression, was associated with higher anastomotic leakage rates. CONCLUSIONS: COX-2-induced PGE2 production is essential for intestinal wound healing after colonic surgery, possibly via its effects on angiogenesis. These data emphasize that COX-2 inhibitors should be avoided after colonic surgery, and administration of PGE2 might be favorable for a selection of patients.

The Human Colon Is More Resistant to Ischemia-reperfusion-induced Tissue Damage Than the Small Intestine: An Observational Study.

OBJECTIVE: Aim of this study was to draw comparisons between human colonic and jejunal ischemia-reperfusion sequelae in a human in vivo experimental model. BACKGROUND: In patients, colonic ischemia-reperfusion generally has a milder course than small intestinal ischemia-reperfusion. It is unclear which pathophysiologic processes are responsible for this difference. METHODS: In 10 patients undergoing colonic surgery and 10 patients undergoing pancreaticoduodenectomy, 6 cm colon or jejunum was isolated and exposed to 60 minutes ischemia followed by various reperfusion periods. Morphology (hematoxylin and eosin), apoptosis (M30), tight junctions (zonula occludens 1), and neutrophil influx (myeloperoxidase) were assessed using immunohistochemistry. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay were performed for interleukin-6 and tumor necrosis factor-α. RESULTS: Hematoxylin and eosin staining revealed intact colonic epithelial lining, but extensive damage in jejunal villus tips after 60 minutes ischemia. After reperfusion, the colonic epithelial lining was not affected, whereas the jejunal epithelium was seriously damaged. Colonic apoptosis was limited to scattered cells in surface epithelium, whereas apoptosis was clearly observed in jejunal villi and crypts, (42 times more M30 positivity compared with colon, P < 0.01). Neutrophil influx and increased tumor necrosis factor-α mRNA expression were observed in jejunum after 30 and 120 minutes of reperfusion (P < 0.05). Interleukin-6 mRNA expression was increased in jejunum after 120 minutes of reperfusion (3.6-fold increase, P < 0.05), whereas interleukin-6 protein expression was increased in both colon (1.5-fold increase, P < 0.05) and small intestine (1.5-fold increase, P < 0.05) after 30 and 120 minutes of reperfusion. CONCLUSIONS: Human colon is less susceptible to IR-induced tissue injury than small intestine.

Complement alternative pathway activation in human nonalcoholic steatohepatitis.

The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (rs = 0.69; p<0.05) and C3c/C3 activation ratio (rs = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway activation in driving hepatic inflammation in NASH. Therefore, alternative pathway factors may be considered attractive targets for treating NASH by inhibiting complement activation.

L-citrulline improves splanchnic perfusion and reduces gut injury during exercise.

PURPOSE: Splanchnic hypoperfusion is a physiological phenomenon during strenuous exercise. It has been associated with gastrointestinal symptoms and intestinal injury and may hamper athletic performance. We hypothesized that L-citrulline supplementation improves splanchnic perfusion and decreases intestinal injury by enhancing arginine availability. The aim of this study was to determine the effect of L-citrulline intake on splanchnic perfusion, intestinal injury, and barrier function during exercise. METHODS: In this randomized, double-blind crossover study, 10 men cycled for 60 min at 70% of their maximum workload after L-citrulline (10 g) or placebo (L-alanine) intake. Splanchnic perfusion was assessed using gastric air tonometry. Sublingual microcirculation was evaluated by sidestream dark field imaging. Plasma amino acid levels and intestinal fatty acid binding protein concentrations, reflecting enterocyte damage, were assessed every 10 min. Urinary excretion of sugar probes was measured to evaluate intestinal permeability changes. RESULTS: Oral L-citrulline supplementation enhanced plasma citrulline (1840.3 ± 142.3 µM) and arginine levels (238.5 ± 9.1 µM) compared with that in placebo (45.7 ± 4.8 µM and 101.5 ± 6.1 µM, respectively, P < 0.0001), resulting in increased arginine availability. Splanchnic hypoperfusion was prevented during exercise after L-citrulline ingestion (reflected by unaltered gapg-apCO2 levels), whereas gapg-apCO2 increased with placebo treatment (P < 0.01). Accordingly, L-citrulline intake resulted in an increased number of perfused small sublingual vessels compared with that in placebo (7.8 ± 6.0 vs -2.0 ± 2.4, P = 0.06). Furthermore, plasma intestinal fatty acid binding protein levels were attenuated during exercise after L-citrulline supplementation compared with that in placebo (AUC0-60 min, -185% ± 506% vs 1318% ± 553%, P < 0.01). No significant differences were observed for intestinal permeability. CONCLUSIONS: Pre-exercise L-citrulline intake preserves splanchnic perfusion and attenuates intestinal injury during exercise in athletes compared with placebo, probably by enhancing arginine availability. These results suggest that oral L-citrulline supplementation is a promising intervention to combat splanchnic hypoperfusion-induced intestinal compromise.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon.

OBJECTIVE: Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. DESIGN: In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1ß and tumour necrosis factor α. RESULTS: In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. CONCLUSIONS: Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

Total parenteral nutrition induces a shift in the Firmicutes to Bacteroidetes ratio in association with Paneth cell activation in rats.

The use of total parenteral nutrition (TPN) in the treatment of critically ill patients has been the subject of debate because it has been associated with disturbances in intestinal homeostasis. Important factors in maintaining intestinal homeostasis are the intestinal microbiota and Paneth cells, which exist in a mutually amendable relationship. We hypothesized that the disturbed intestinal homeostasis in TPN-fed individuals results from an interplay between a shift in microbiota composition and alterations in Paneth cells. We studied the microbiota composition and expression of Paneth cell antimicrobial proteins in rats receiving TPN or a control diet for 3, 7, or 14 d. qPCR analysis of DNA extracts from small intestinal luminal contents of TPN-fed rats showed a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes after 14 d (P < 0.05) compared with the control group. This finding coincided with greater staining intensity for lysozyme and significantly greater mRNA expression of the Paneth cell antimicrobial proteins lysozyme (P < 0.05), rat α-defensin 5 (P < 0.01), and rat α-defensin 8 (P < 0.01). Finally, 14 d of TPN resulted in greater circulating ileal lipid-binding protein concentrations (P < 0.05) and greater leakage of horseradish peroxidase (P < 0.01), which is indicative of enterocyte damage and a breached intestinal barrier. Our findings show a shift in intestinal microbiota in TPN-fed rats that correlated with changes in Paneth cell lysozyme expression (r(s) = -0.75, P < 0.01). Further studies that include interventions with microbiota or nutrients that modulate them may yield information on the involvement of the microbiota and Paneth cells in TPN-associated intestinal compromise.

Aggravation of exercise-induced intestinal injury by Ibuprofen in athletes.

INTRODUCTION: Nonsteroidal anti-inflammatory drugs are commonly used by athletes to prevent anticipated exercise-induced pain, thereby putatively improving physical performance. However, these drugs may have potentially hazardous effects on the gastrointestinal (GI) mucosa during strenuous physical exercise. The aim of the current study was to determine the effect of oral ibuprofen administration before exercise on GI integrity and barrier function in healthy individuals. METHODS: Nine healthy, trained men were studied on four different occasions: 1) 400 mg ibuprofen twice before cycling, 2) cycling without ibuprofen, 3) 400 mg ibuprofen twice at rest, and 4) rest without ibuprofen intake. To assess small intestinal injury, plasma intestinal fatty acid binding protein (I-FABP) levels were determined, whereas urinary excretion of orally ingested multisugar test probes was measured using liquid chromatography and mass spectrometry to assess GI permeability. RESULTS: Both ibuprofen consumption and cycling resulted in increased I-FABP levels, reflecting small intestinal injury. Levels were higher after cycling with ibuprofen than after cycling without ibuprofen, rest with ibuprofen, or rest without ibuprofen (peak I-FABP, 875 ± 137, 474 ± 74, 507 ± 103, and 352 ± 44 pg·mL, respectively, P < 0.002). In line, small intestinal permeability increased, especially after cycling with ibuprofen (0-2 h urinary lactulose/rhamnose ratio, 0.08 (0.04-0.56) compared with 0.04 (0.00-0.20), 0.05 (0.01-0.07), and 0.01 (0.01-0.03), respectively), reflecting loss of gut barrier integrity. Interestingly, the extent of intestinal injury and barrier dysfunction correlated significantly (RS = 0.56, P < 0.001). CONCLUSION: This is the first study to reveal that ibuprofen aggravates exercise-induced small intestinal injury and induces gut barrier dysfunction in healthy individuals. We conclude that nonsteroidal anti-inflammatory drugs consumption by athletes is not harmless and should be discouraged.

Thromboxane receptor signalling in renal ischemia reperfusion injury.

F(2)-isoprostanes are formed by oxidative modification of arachidonic acid and are the gold standard for detection of oxidative stress in vivo. F(2)-isoprostanes are biologically active compounds that signal through the thromboxane A(2) (TP) receptor; infusion of F(2)-isoprostanes reduces glomerular filtration in the kidney by constricting afferent arterioles. This study investigated whether endogenous F(2)-isoprostanes contribute to the pathogenesis of ischemic acute kidney injury, which is associated with oxidative stress and reduced glomerular filtration. TP receptor knockout mice-that lack F(2)-isoprostanes and thromboxane A(2) signalling-and wild-type control mice underwent 30 min of renal ischemia and 24 h of reperfusion. Kidney dysfunction, histological injury and the number of infiltrated neutrophils were similar between the two mouse strains, whereas TP receptor knockout mice had significantly more apoptotic cells and tissue lipid peroxidation than their wild-type counterparts. F(2)-isoprostanes and thromboxane B(2) were readily detectable in urine collections after surgery. The findings indicate that F(2)-isoprostanes and thromboxane A(2) signalling do not contribute critically to the pathogenesis of ischemic acute kidney injury and more generally provide evidence against a prominent role for F(2)-isoprostanes signalling in exacerbating acute disease states associated with oxidative stress.

Bioaccumulation of elements in bryophytes from Serra da Estrela, Portugal, and Veluwezoom, the Netherlands.

BACKGROUND, AIMS AND SCOPE: Pollution by heavy metals over large areas and long periods of time may cause chronic damage to living organisms and must be carefully controlled. One way to determine the extent of environmental contamination is by measuring the levels of contaminants in plants. The use of mosses as biomonitors is a convenient method to determine levels of (atmospheric) deposition, as terrestrial mosses obtain most of their supply of mineral elements from precipitation and dry deposition of airborne particles. Mosses have therefore received increasing attention as a suitable tool for monitoring regional patterns of elemental deposition from the atmosphere in large-scale studies in various countries, in areas close to industrial installations as well as in areas not expected to be contaminated. Although this technique is widely known, ecological studies of this type have rarely been done in Portugal. The aim of this paper is to evaluate and compare the spatial distribution of heavy metals in Hypnum cupressiforme, Pleurozium schreberi, Dicranum scoparium and Polytrichum piliferum collected from the Serra da Estrela natural park in Portugal and in the Veluwezoom natural park in the Netherlands. The selected species are the most widely used bryophytes for biomonitoring in the boreal region. The popularity of these species for this purpose is due to their wide ecological amplitude and distribution. METHODS: At 54 sampling sites in both nature parks, samples of Hypnum cupressiforme, Pleurozium schreberi, Dicranum scoparium and Polytrichum piliferum were collected. Plant digests were analysed for Al, Ba, Ca, Cr, Fe, K, Mg, Mn, Ni, Sr, V, Zn, Pb, Cu, Cd, N and P. Differentiations between sampling sites in terms of concentrations of elements in mosses were evaluated by ANOVA and the least significant difference was calculated. The normality of the analysed features was checked with the chi square test. After standardization, the matrix of 54 samples and 10 heavy metals was subjected to numerical classification to detect groups of samples with similar patterns of metal concentrations. The clustering algorithm was prepared with Ward's method, and the City Block Manhattan method was used for the similarity measure. Metals and samples were also subjected to ordination to reveal possible gradients of heavy metal levels, using PCA. Correlations were calculated between concentrations of metals and factors 1 and 2, allowing the dependence between the concentration of metals and factors (factor loading) to be estimated. RESULTS AND DISCUSSION: All species examined in both areas contained elevated levels of Mn and Pb. For each particular species, concentrations of N, P and Pb were significantly higher at Serra da Estrela, while concentrations of Cu were significantly higher at the Veluwezoom. Mosses from Portugal and the Netherlands differed significantly mainly in the concentrations of Al, Ba, Cr, Fe, Mn, Ni, Pb and V. This differentiation did not exceed that within the mosses from Portugal. CONCLUSIONS: Mosses from Portugal and the Netherlands differ significantly mainly in the concentrations of Al, Ba, Cr, Fe, Mn, Ni, Pb and V. This differentiation does not exceed the differentiation within the mosses from Portugal. RECOMMENDATION AND OUTLOOK: Further research is required into the origin and deposition of the polluting elements in other environmental compartments.