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The use of machine perfusion (MP) in liver transplantation (LT) is spreading worldwide. However, its efficacy has not been demonstrated, and its proper clinical use has far to go to be widely implemented. The Società Italiana Trapianti d’Organo (SITO) promoted the development of an evidence-based position paper. A 3-step approach has been adopted to develop this position paper. First, SITO appointed a chair and a cochair who then assembled a working group with specific experience of MP in LT. The Guideline Development Group framed the clinical questions into a patient, intervention, control, and outcome (PICO) format, extracted and analyzed the available literature, ranked the quality of the evidence, and prepared and graded the recommendations. Recommendations were then discussed by all the members of the SITO and were voted on via the Delphi method by an institutional review board. Finally, they were evaluated and scored by a panel of external reviewers. All available literature was analyzed, and its quality was ranked. A total of 18 recommendations regarding the use and the efficacy of ex situ hypothermic and normothermic machine perfusion and sequential normothermic regional perfusion and ex situ MP were prepared and graded according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method. A critical and scientific approach is required for the safe implementation of this new technology.

The pervasive shortage of deceased donor liver allografts contributes to significant waitlist mortality despite efforts to increase organ donation. Ex vivo liver perfusion appears to enhance preservation of donor organs, extending viability and potentially evaluating function in organs previously considered too high risk for transplant. These devices pose novel challenges for organ allocation, safety, training, and finances. This white paper describes the American Society of Transplant Surgeons' belief that organ preservation technology is a vital advance, but its use should not change fundamental aspects of organ allocation. Additional data elements need to be collected, made available for organ assessment by transplant professionals to allow determination of organ suitability in the case of reallocation and incorporated into risk adjustment methodology. Finally, further work is needed to determine the optimal strategy for management and oversight of perfused organs prior to transplantation.

In this paper, we propose a generalized concept of microfluidic polydimethylsiloxane (PDMS) on paper (POP) devices, which combines well the merits of paper chips and PDMS chips. First, we optimized the conditions for accurate PDMS spatial patterning on paper, based on screen printing and a high temperature enabled superfast curing technique, which enables PDMS patterning to an accuracy of tens of microns in less than ten seconds. This, in turn, makes it available for seamless, reversible and reliable integration of the resulting paper layer with other PDMS channel structures. The integrated POP devices allow for both porous paper and smooth channels to be spatially defined on the devices, greatly extending the flexibility for designers to be able to construct powerful functional structures. To demonstrate the versatility of this design, a prototype POP device for the colorimetric analysis of liver function markers, serum protein, alkaline phosphatase (ALP) and aspartate aminotransferase (AST), was constructed. On this POP device, quantitative sample loading, mixing and multiplex analysis have all been realized.

In this work, a novel calibration approach for minor and trace element quantification in LA-ICP-MS imaging of biological tissues is presented. Droplets of aqueous standard solutions are deposited onto pre-cut pieces of filter paper, allowed to dry, and sputtered with a thin gold layer for use as pseudo-internal standard. Analysis of the standards using LA-ICP-MS is performed using radial line-scans across the filters. In contrast to conventionally used preparation of matrix-matched tissue standards, the dried-droplet approach offers a variety of advantages: The standards are easy to prepare, no characterization of the standards using acid digestion is required, no handling of biological materials is necessary, and the concentration range, as well the number of investigated analytes is almost unlimited. The proposed quantification method has been verified using homogenized tissue standards with known analyte concentrations before being applied to a human malignant mesothelioma biopsy from a patient who had not received any chemotherapeutic treatment. Elemental distribution images were acquired at a lateral resolution of 40 μm per pixel, limits of detection ranging from 0.1 μg g(-1) (Mn, Ni, Cu, Zn) to 13.2 μg g(-1) (K) were reached.

Pulp and paper mill effluents (PPMEs) have been shown to increase gonad size, cause early maturation, and disrupt hormone functions in native and non-native Chilean fish. In this study, we assessed reproductive (plasma vitellogenin; VTG, gonad development) and metabolic (ethoxyresorufin-. O-deethylase activity; EROD) end points, relative liver size (LSI) and condition factor (K) of juvenile female and male rainbow trout exposed to effluents. Unlike previous studies, which have focus either on the specific effects of effluent on fish in laboratory exposures or biotic population statuses downstream of discharge sites, we simultaneously assessed the impacts of PPMES on trout using two approaches: (1) laboratory exposures of tertiary treated PPME produced from processing Eucalyptus globulus or Pinus radiata; and (2) in situ bioassay downstream of the combined discharge of the same pulp mill. Despite an increase in the average gonadosomatic index (GSI) in exposed fish, no statistical differences in gonad size between exposed and unexposed individuals was detected. However, both female and male fish exposed to effluents showed significantly higher concentrations of plasma VTG, so more in fish exposed to Eucalyptus-based effluent when compared to Pinus PPME. In addition, male fish showed intersex characteristics in all exposure assays (Eucaliptus and Pinus) and, despite the low concentration of effluent in the river (

Bioactive substances in the Biobio River Basin in Chile were examined by deploying Semi Permeable Membrane Devices (SPMDs) upstream and downstream of 4 pulp mill effluent discharges. Androgenic and estrogenic activity of SPMD extracts were then evaluated using in vitro fish sex steroid receptor binding assays. The results indicated the occurrence of estrogenic type compounds associated with one of the mill discharges. A significant correlation among the presence of these compounds, an increase in gonadosomatic index GSI and induction of hepatic EROD activity of two native fish species was observed. However, no significant presence of mature oocytes in female gonads was detected. Although EROD induction was observed in sites impacted by mill effluents, an increase of its activity occurred towards the downstream areas, suggesting other non-mill sources. More research is needed to understand the environmental changes in context of the new technological improvements in treatment systems to MBBR (Moving Bed Biofilm Reactor) recently implemented by the pulp mill industries.

We report here on studies designed to asses the effects of paper mill effluents on non-reproductive functions of free-ranging and captive Florida largemouth bass (Micropterus salmoides floridanus). This was accomplished by conducting an outdoor tank study, in which fish were exposed to well water or to 10%, 20%, 40%, and 80% full strength effluent for 28 or 56 days, and by sampling largemouth bass from sites within the St. Johns River, Florida, upstream and downstream from a paper mill plant. Blood and plasma samples from fish from the tank study and from fish sampled from the ambient sites were analyzed for over 20 variables. We also determined liver and spleen weights and examined them histologically. The most significant finding from the tank study was an increase in the concentration of albumin and hepatosomatic index for bass exposed toor = 20% effluents for 56 days. Spleenosomatic index and number of melanomacrophage centers were decreased in bass from effluent-dominated sites (Palatka and Rice Creek), whereas concentrations of calcium, phosphorous, glucose, and creatinine were elevated in fish from these sites, compared to fish from reference streams. Fish from Rice Creek also had fewer red blood cells, and male bass from Palatka had lower concentrations of cholesterol. Plasma concentrations of albumin and hepatic concentrations of glutathione were elevated in males from Palatka, and both females and males from Rice Creek had higher concentrations of globulin. These results indicate a complex pattern of effects of paper mill effluents on several physiological functions. However, despite the myriad of treatment and site-related effects, most physiological parameters fell within normal ranges when compared to reports on largemouth bass and other freshwater species.

While recent evidence indicates that the generation of reactive oxygen species (ROS) and associated oxidative damage are frequently observed in fish populations with exposure to pulp and paper mill effluents, the potential for ROS generation from municipal sewage effluents has not been addressed. This study investigates the utility of measures of oxidative stress in delineating the effects of both pulp and paper mill and municipal sewage discharges. Longnose sucker (Catostomus catostomus) were collected below three pulp and paper mill and two municipal sewage effluent discharges over a 3-year period within the Wapiti and Athabasca River systems in northern Alberta. Biochemical responses in longnose sucker varied between the two rivers systems, with more pronounced changes occurring within the Wapiti River. Of the suite of biochemical parameters examined, fatty acyl-CoA oxidase (FAO) activity was the most sensitive indicator of pulp and paper mill exposure, but was only infrequently induced with exposure to municipal sewage effluent. Hepatic and gonadal 2-thiobarbituric acid reactive substances, lipid hydroperoxides, and hepatic free iron were less consistently elevated with exposure to pulp and paper mill effluent than FAO activity, and were also only infrequently altered with sewage effluent exposure. Hepatic ascorbic acid, liver somatic index, and condition factor were consistently altered with exposure to both sewage and pulp and paper mill effluents. While specific biochemical and organismal responses varied with effluents and time, the collective suite of oxidative stress endpoints proved to be useful tools in identifying relative influences of municipal sewage and pulp and paper mill effluent on fish populations in adjacent receiving waters.

Diacetyl (2,3-butanedione) is a yellowish liquid that is usually mixed with other ingredients to produce butter flavor or other flavors in a variety of food products. Inhalation of butter flavoring vapors was first associated with clinical bronchiolitis obliterans among workers in microwave popcorn production. Recent findings have shown irreversible obstructive lung disease among workers not only in the microwave popcorn industry, but also in flavoring manufacture, and in chemical synthesis of diacetyl, a predominant chemical for butter flavoring. It has been reported that perfluorochemicals utilized in food packaging are migrating into foods and may be sources of oral exposure. Relatively small quantities of perfluorochemicals are used in the manufacturing of paper or paperboard that is in direct contact with food to repel oil or grease and water. Because of recent concerns about perfluorochemicals such as those found on microwave popcorn bags (e.g. Lodyne P208E®) and diacetyl in foods, we evaluated both compounds for mutagenicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells. Lodyne P208E® was less toxic than diacetyl and did not induce a mutagenic response. Diacetyl induced a highly mutagenic response in the L5178Y mouse lymphoma mutation assay in the presence of human liver S9 for activation. The increase in the frequency of small colonies in the assay with diacetyl indicates that diacetyl causes damage to multiple loci on chromosome 11 in addition to functional loss of the thymidine kinase locus.

Female mosquitofish exposed to pulp and paper mill effluent (PME) in the Fenholloway River, Florida, USA have masculinized secondary sex characteristics and altered aromatase enzyme activity. We and others have shown that the Fenholloway River PME contains androgenic and progestogenic substance(s). The present study was designed to test the hypothesis that the development and reproductive health of PME-exposed Fenholloway River mosquitofish are altered compared to mosquitofish living in Econfina River, which is the reference site. Fish were collected on a single day from both sites in June and August 1999 and January and June 2000. We compared standard length, anal fin length and segment number; body, liver, and gonad mass; and number of eggs and embryos from Fenholloway and Econfina River mosquitofish. The data were analyzed collectively for generalized site effect, for site effects during reproductive and nonreproductive seasons, and for repeatability of site effects between years. Mosquitofish exposed to PME in the Fenholloway River were generally smaller in length and mass, anal fin segment number was greater, and the number of embryos, but not oocytes, was significantly decreased compared to the reference site fish. Anal fin length and segment number and liver and testis masses were generally greater in Fenholloway compared to the Econfina River males. The importance of this study is that we have documented masculinized development and decreased embryo production in PME-exposed mosquitofish and that these site effects are generally consistent across seasons and between years.

Several environmental contaminants have been found in paper mill effluent including substances with androgenic properties. The presence of androgenic substances in paper mill effluent was also indicated in the present study which demonstrated masculinized anal fins of female mosquitofish from the paper mill effluent contaminated Fenholloway River. In addition, when compared to the nearby Econfina River, which does not receive paper mill effluent, fewer females from the contaminated river were pregnant, they were smaller and their estradiol concentration exhibited greater variation. Males from the Fenholloway River and the Econfina River had similar sperm counts, but the testes were larger and greater variation in testosterone concentration was observed in male fish from the Fenholloway River. In males and females from the Fenholloway River, liver weights were increased and computer-aided behavior analysis demonstrated a reduction in their social behavior when compared to reference fish. We conclude that a number of sexual characteristics were affected in mosquitofish living in the paper mill contaminated Fenholloway River, with possible adverse effects on the reproduction of this population.

Laboratory tests have been used to assess the regulatory and research questions related to the effects of pulp mill effluents on aquatic biota. Acute, short-term laboratory tests have clearly shown the improvement in final effluent quality following installation of secondary treatment at Canadian pulp mills. In an effort to predict and investigate impacts on wild fish, laboratory bioassays were developed to examine sublethal endpoints: induction of hepatic mixed function oxygenase activity and reduction of sex steroid concentrations. These laboratory assays have been used to assess whole effluents, specific chemicals, and components of pulp mill processes, and to discriminate between historical and present-day effluent discharges. These tests have shown that induction of mixed-function oxygenase activity and reduction of sex steroid concentrations are produced by effluents from a variety of mill types, with and without chlorine bleaching, in hardwood and softwood pulping facilities, and before and after effluent treatment. These short-term bioassays have enabled reductions in sex steroid concentrations to be linked to mill process streams, and have provided information on effective waste stream treatment. Longer term, life-cycle fish bioassays have shown that chronic exposure to pulp mill effluents commonly results in growth enhancement, liver enlargement, and decreases in gonad size, secondary sex characteristics, and fecundity. These long-term laboratory exposures are able to mimic the most commonly observed alterations of wild fish exposed to pulp mill effluents: increases in condition factors, increases in liver-somatic indices, and decreases in gonadosomatic indices. This pattern of response is a combination of nutrient enrichment with metabolic disruption. The most sensitive and biologically meaningful endpoint is decreased reproduction in fish life-cycle exposures. As the laboratory tests move forward into the next decade, attention will focus on the reproductive endpoints and on the possibility of shortening the fish bioassays while still maintaining sensitivity and biological relevance.

Liver biopsy (LB) is still the criterion standard procedure for obtaining liver tissue for histopathological examination and a valuable tool in the diagnosis, prognosis, and management of many parenchymal liver diseases. The aim of this position paper is to summarise the present practice of paediatric LB and make recommendations about its performance. Although histological evaluation of the liver is important in assessing prognosis and exploring treatment, noninvasive techniques (ie, imaging, laboratory markers) may replace use of liver histology. The indications for LB are changing as present knowledge of aetiologies, pathomechanism, and therapeutic options in paediatric liver disease is evolving. Adult and paediatric literature was reviewed to assess the existing clinical practice of LB with focus on the technique, indications, risk of complications, and contraindications in paediatrics. This position paper presents types of LB, indications, complications, contraindications, and an essential checklist for paediatric LB.

To develop an effective hybrid bioartificial liver (BAL) device, the material of the scaffold is very important to support hepatocytes that have both growth ability and hepatic differentiated functions. In this study we used paper (Kimwipe, Kimberly-Clark Corp., Roswell, GA, U.S.A.) as a scaffold. Primary hepatocytes isolated from a normal adult rat liver could proliferate on the paper. The secretion of albumin into culture medium by the cells on the paper increased with time in culture and, compared to the cells on dishes, the amount of 48 h albumin secretion at Day 10 was two times larger. Perpendicular sections of hepatocytes on the paper revealed that the cells fell into cavities made by intersecting fibers, piled up, and formed three to four layers. The piled-up cells changed their shape from flat to cuboidal and enlarged their cytoplasm, which was rich in organelles such as mitochondria and peroxisomes with a nucleoid. In addition, they formed bile canalicular structures between the cells. Their morphological appearance was similar to in vivo hepatocytes. Paper (Kimwipe) may be a good candidate as a scaffold to make a BAL device.

Valnemulin (VAL) and tiamulin (TIA) are pleuromutilin antibiotics used primarily for treating bacterial infections in swine or other food animals. Furthermore, VAL and TIA are also employed as feed additives to promote animal growth. However, the illegal use of VAL and TIA could cause a series of hazards to consumers. Here, VAL was designed to be conjugated with bovine serum protein to prepare immunogen. A highly sensitive monoclonal antibody that recognized both VAL and TIA has been successfully produced. Moreover, an immunochromatographic strip assay for rapidly screening VAL and TIA in porcine liver was established with visual detection limits (cutoff values) of 50 and 25 ng/g, respectively. The IC50 values calculated from the equation of the standard curve were 6.06 and 3.45 ng/g and the limits of detection were 0.96 and 0.29 ng/g for VAL and TIA. According to the recovery experiment results, the test strip exhibited acceptable accuracy and precision. Generally, the proposed strip provided a practical tool for the detection of VAL and TIA. PRACTICAL APPLICATION: We produced a highly sensitive monoclonal antibody and developed an immunoassay strip for simultaneously monitoring TIA and VAL. Additionally it was preliminarily confirmed that the rapid detection tool was suitable for screening TIA and VAL in porcine liver.

Physiological and biochemical biomarker responses were studied in juvenile whitefish (Coregonus lavaretus L. s.l.) exposed experimentally to effluent from the forest industry. The large study area (609 km2), Southern Lake Saimaa, in Southeast Finland, receives 330,000 m3 d-1 of biologically and 55,000 m3 d-1 of chemically treated effluents, discharged from two integrated elementary chlorine free (ECF) bleached kraft pulp and paper mills, from one ECF pulp mill, and from one mill producing unbleached pulp and cardboard. The assessment of exposure to effluent discharged from the mills was based on lake water chlorophenolics (CPs) and resin acids (RAs) measured in samples collected from the 22 experimental sites along the area. Despite the low levels of effluent constituents in the lake, they were still accumulated in detectable levels in fish bile, indicating an exposure to the bioactive compounds of effluents. In comparison to the reference area, a two- to four-fold increase in ethoxyresorufin O-deethylase (EROD) activity was observed in whitefish exposed in the vicinity (1-6 km) of all the mills. However, cytochrome P450 1A1 (CYP1A1) gene expression was increased in only one of the receiving areas, indicating higher sensitivity of the EROD activity in the present study. There were no statistically significant correlations between EROD activity and the ambient water concentrations of the CPs, the RAs, or effluent dilution expressed by water sodium concentration. Neither bile chlorophenolics nor bile resin acids showed a significant correlation with EROD. No significant changes in circulating reproductive steroids, 17beta-estradiol and testosterone, in juvenile whitefish were observed. The vitellogenin gene was expressed in the vicinity of the pulp mill discharging the most wood-derived compounds, i.e. resin acids and wood-sterols, including beta-sitosterol. No differences were observed in plasma immunoglobulin M, glucose, or lactate concentrations between the effluent sources.

Juvenile whitefish (Coregonus lavaretus L. s.l.) were exposed by caging in the field to diluted effluents from three operating pulp, paper, and paperboard mills in Southern Lake Saimaa, Finland. The expression of the vitellogenin gene, used as a biomarker of estrogenic contamination of effluents, was measured using a Northern blotting method. Increased mRNA levels, the most specific and reliable evidence for estrogen receptor-mediated actions in vivo, were found in fish caged in the vicinity of one of three mills studied. This mill was found to discharge wood-derived compounds, such as sterols and resin acids, into Lake Saimaa in amounts considerably exceeding those from the other two mills. The increased vitellogenin gene expression suggests that the effluent is a source of estrogenic contaminants.

This study investigates whether pulse exposure to Chilean pulp and paper mill effluent solid phase extracted (SPE) extracts via intraperitoneal injection (IP), would result in changes in the activities of the respiratory metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) in rainbow trout livers. It also investigated if an alteration in liver metabolic capacity influenced the liver detoxification processes and estrogenic effects previously reported. Besides, a comparison of those enzymatic activities with fish IP injected with SPE extracts of two model effluents coming from industries that process 100% different type of feedstock (softwood, SW and hardwood, HW) was also evaluated. An initial induction of the anaerobic metabolism (increase in LDH enzymatic activity) was detected in all Chilean pulp mill effluent extracts evaluated, contrary to the initial unaltered aerobic metabolism (CS enzymatic activity) observed. A compensatory relationship in energy metabolism (Pasteur effect) was observed when comparing both enzymatic activities of fish exposed to those effluent extracts. LDH and CS activities observed in fish injected with Chilean extracts seem to be related to the effects observed in fish injected with SW extracts. This study showed that intraperitoneal injection of pulp and paper mill effluent extracts affected the anaerobic and aerobic metabolic capacities in rainbow trout livers, but this metabolic alteration did not affect detoxification capability or estrogenic effect previously reported.

In order to validate previous field observations by the authors on whitefish, Coregonus lavaretus L. s.l., a 30-day laboratory experiment with concentrations (0, 1.3, 2.3, 3.5, and 7 vol%) of bleached kraft pulp and paper mill effluent (BKME) simulating those occurring in a polluted lake was conducted. Chlorine dioxide had almost entirely replaced chlorine gas in the bleaching of pulp. As a consequence, the concentrations of adsorbable organic halogens and chlorinated phenolics (CPs) in BKME were significantly lowered compared to earlier studies. This reduction was also seen in the concentrations of CPs in the bile and CPs and extractable organic halogens in the intestinal lipids: the concentrations were low and did not depend on the dilution of BKME. In contrast, the resin acid content of bile decreased with decreasing BKME concentration. The growth of fish was speeded up in all BKME concentrations. However, at the highest BKME concentration (7 vol%) the increase was lowest. The induction of hepatic ethoxyresorufin O-deethylase (EROD) activity revealed strong dose-response relationship with BKME. At 3.5 vol% BKME (corresponding to a distance of 3.3 km from the mill sewer in the field) the EROD activity increased 12-fold. There was a tendency for lower activity of uridinediphosphate glucuronosyltransferase in the liver, but the decrease (34%; P < 0.05) was statistically significant only at 7 vol% BKME. The activity of liver glutathione S-transferase remained unchanged. All dilutions of BKME significantly depressed the concentrations of plasma immunoglobulin M (IgM). Erythrocytic concentrations of nucleotide triphosphates decreased and of sodium increased as the BKME concentration increased. Also some other blood parameters (hematocrit, hemoglobin, plasma glucose, and aspartate aminotransferase) were changed in all BKME exposures, although without obvious dependence on effluent concentration. In conclusion, there was a good agreement between field studies and laboratory experiments using BKME concentrations occurring in the field, confirming close or similar causes for responsive toxicity endpoints.

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and “spiked” standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.

The purpose of this study was to evaluate the effects of bleached kraft mill effluent on selected physiological and hematological endpoints in fingerling largemouth bass (Micropterus salmoides). Fish were exposed for 35 days to surface water collected at sites up- and downstream from a pulp and paper mill treatment-effluent discharge. The condition factor, spleen somatic index, and pronephros cellularity were statistically significantly decreased in fish exposed to the downstream site, compared to the upstream site. There was also a shift from lymphocytes to neutrophils in peripheral blood of fish exposed to the downstream site. Hematocrit values and the liver somatic index were statistically significantly increased in fish in the downstream exposure. These results indicate that in-stream exposure to elemental-chlorine-free pulp and paper mill effluents produce a generalized stress response, leading to potential immunosuppression in fish.

Eight phenolics were detected in samples collected from areas where paper-recycling process water is discharged. The detected concentration levels were up to 270 microg/L and 230 microg/g in water samples and sediment samples, respectively, obtained from both the outfall of the paper-recycling process water and its downstream areas. In particular, totarol (compound 4), 2,4-bis(1-phenylethyl)phenol (compound 6), 4,4'-butylidenebis(6-t-butyl-m-cresol) (compound 7), 2,4-bis(1-phenylethyl)-6-chlorophenol (compound 8), and 4-hydroxy-4'-isopropoxydiphenyl sulfone (compound 9) were identified for the first time as environmental pollutants. The estrogenicities of the identified compounds were assessed by yeast two-hybrid assays incorporating either the human or medaka fish (Oryzias latipes) estrogen receptor alpha (hERalpha and medERalpha, respectively) and an estrogen receptor competitive enzyme-linked immunosorbent assay (ER-ELISA) both with and without metabolic activation by a rat liver S9 mix. Bisphenol A (compound 3) and 2-naphthol (compound 1) exhibited activity in the assays of both hERalpha and medERalpha without the S9 mix. The relative activity (%) to 17beta-estradiol was 0.0015% for compound 3 and 0.0009% for compound 1 in the hERalpha assay and 0.027% for compound 3 and 0.0093% for compound 1 in the medERalpha assay. These compounds were attenuated by the S9 mix. The binding affinity was evaluated using an ER-ELISA. Compounds 3, 4, 6, and 7 exhibited affinity without the S9 mix. After exposure to the S9 mix, however, the binding affinity of compound 7 was eliminated by the S9 mix; those of compounds 3, 4, and 6 were attenuated; and that of compound 8 exhibited affinity. A comprehensive assessment of the estrogenicities of the phenolics originating from thermal paper and their implications for an aquatic environment may require an examination of the components of the phenolics, as in the present study.

Two experiments using rainbow trout (Oncorhynchus mykiss) were conducted to examine the combined effects of energy intake as manipulated by ration and pulp and paper mill effluent exposure over either one, or two consecutive reproductive cycles. This study demonstrated that the level of energy intake affected the full range of measured parameters from energy allocation to somatic growth and the gonadal development, steroid production and hematology. Increasing ration level expectedly increased growth, condition, liver and gonad size. Female trout in the higher ration treatments produced more follicles and had larger eggs, investing the same relative proportion of total energy into ovarian development. Sex steroid levels and hematological parameters were also positively influenced by increasing ration level in males and females. By far, the most dramatic impact of reduced ration on reproduction was to substantially reduce the frequency of sexually maturing fish. The effects of effluent exposure were not as marked as those linked to ration level and typically did not manifest unless fish were exposed through two consecutive reproductive cycles. The physiological effects of pulp and paper effluent exposure observed in these experiments were not consistent between the two experiments conducted herein, nor were they consistent with previously observed impacts in similar experiments with this effluent. Effluent exposure over one reproductive cycle did not impact physiological parameters in trout. However, when effluent exposure was maintained over two reproductive cycles, a new pattern of effluent response emerged including increased condition factor in both sexes, a decrease in the potential ability of the blood of females to transport oxygen, and increased sex steroids and reproductive investment in males. Effluent was also observed to cause reduced growth in male trout over two years. The effects of ration on gonad and liver size were far more obvious and consistent when a longer exposure was employed, thus, it appears to take more than one full year for energy intake changes to be reflected in those particular physiological endpoints.

Many studies link pulp and paper mill effluent (PPME) exposure to adverse effects in fish populations present in the mill receiving environments. These impacts are often characteristic of endocrine disruption and may include impaired reproduction, development and survival. While these physiological endpoints are well-characterized, the molecular mechanisms causing them are not yet understood. To investigate changes in gene transcription induced by exposure to a PPME at several stages of treatment, male and female fathead minnows (FHMs) were exposed for 6 days to 25% (v/v) secondary (biologically) treated kraft effluent (TK) or 100% (v/v) combined mill outfall (CMO) from a mill producing both kraft pulp and newsprint. The gene expression changes in the livers of these fish were analyzed using a 22 K oligonucleotide microarray. Exposure to TK or CMO resulted in significant changes in the expression levels of 105 and 238 targets in male FHMs and 296 and 133 targets in females, respectively. Targets were then functionally analyzed using gene ontology tools to identify the biological processes in fish hepatocytes that were affected by exposure to PPME after its secondary treatment. Proteolysis was affected in female FHMs exposed to both TK and CMO. In male FHMs, no processes were affected by TK exposure, while sterol, isoprenoid, steroid and cholesterol biosynthesis and electron transport were up-regulated by CMO exposure. The results presented in this study indicate that short-term exposure to PPMEs affects the expression of reproduction-related genes in the livers of both male and female FHMs, and that secondary treatment of PPMEs may not neutralize all of their metabolic effects in fish. Gene ontology analysis of microarray data may enable identification of biological processes altered by toxicant exposure and thus provide an additional tool for monitoring the impact of PPMEs on fish populations.

Effluents from pulp and paper mills that historically have used elemental chlorine in the bleaching process have been implicated in inhibiting reproduction in fish. Compounds with estrogenic and androgenic binding affinities have been found in these effluents, suggesting that the impairment of reproduction is through an endocrine-related mode of action. To date, a great deal of attention has been paid to phytoestrogens and resin acids that are present in mill process streams as a result of pulping trees. Estrogen and estrogen mimics interact directly with the estrogen receptor and have near immediate effects on gene transcription by turning on the expression of a unique set of genes. Using differential display (DD) RT-PCR, we examined changes in gene expression induced by exposure to paper mill effluents. Largemouth bass were exposed to 0, 10, 20, 40, and 80% paper mill effluent concentrations in large flow-through tanks for varied periods of time including 7, 28 or 56 days. Plasma hormone levels in males and females and plasma vitellogenin (Vtg) in females decreased with dose and time. Measurements of changes in gene expression using DD RT-PCR suggest that the gene expression patterns of male fish do not change much with exposure, except for the induction of a few genes including CYP 1A, a protein that is induced through the action of the Ah receptor in response to dioxin and similar polyaromatic hydrocarbons. However, in the case of females, exposure to these effluents resulted in an up-regulation of CYP 1A that was accompanied by a generalized down-regulation of genes normally expressed during the reproductive season. These antiestrogenic changes are in agreement with previous studies in bass exposed to these effluents, and could result in decreased reproductive success in affected populations.

The responses of shortfin eel (Anguilla australis) to discharges from two pulp and paper mills, municipal wastewater, and a geothermal power plant wastewater were examined. Eels were caged at 3 sites along the Tarawera River, North Island, New Zealand, to explore effects of a 3-wk exposure down a contamination gradient (Ref --D1 --D2). Most of the observed effects were seen in eels caged at the furthest downstream site (D2), below all the discharge areas. General hematology in eels was unaffected, as measures did not differ markedly at the two downstream sites compared with the reference site. At D2, eels were significantly lighter per unit length (reduced condition factor), although liver and spleen size (LSI and SSI) were unaffected. Significantly elevated circulating sex steroid concentrations (testosterone and estradiol) were measured in D2 eels and increasing sex steroid levels at both sites downstream of the reference site were observed. Significant ethoxyresorufin O-deethylase (EROD) activity induction was seen in D2 eels and bile chemistry showed significant accumulation of pyrene and retene equivalents. However, significantly greater concentrations of total resin acids were found in the bile of eels from the intermediate site (D1), between the two pulp and paper mills. The higher polycyclic aromatic hydrocarbon (PAH) equivalents found in the bile of D2 eels suggest that resin acid neutrals, particularly retene, are responsible for some of the effects observed in eels at the furthest downstream exposure site. Levels of pulp and paper mill extractives in sediment, including the PAH retene, support this conclusion.

This study was conducted to ascertain the influence of sediment contaminated with pulp and paper mill effluent in a fjord on winter flounder, Pleuronectes americanus, based on a laboratory study. Flounder, captured from a pristine site, were exposed in a flow-through system for 16 weeks to sediment collected at 2, 5, 7, and 10 km from the outfall. A group of controls was placed in uncontaminated sediment. Mortality occurred almost exclusively in fish exposed to sediment taken from 2 km than from more distant sites. Additionally, the condition factor was lower, the liver was enlarged, and toxicopathic lesions in the liver and spleen were significantly greater in fish submerged in the sediment than in fish from the more distant locations or the controls. Two ectoparasites including a ciliate, Trichodina jadranica, and a monogenean, Gyrodactylus pleuronecti, were observed only in the control group, while a digenean in the digestive tract, Steringophorus furciger, was more abundant in fish exposed to sediment from sites more distant from the outfall and the controls than at 2 km. Comparison of these results with data from a previous gradient field study on biological variables in winter flounder, captured at 2, 5, 7, and 10 km down-current from the outfall, revealed an enlarged liver that was associated with elevated levels of detoxification of hepatic enzymes and prevalence of toxicopathic lesions in both the liver and the spleen; these were significantly greater in samples taken nearest to the outfall from the mill than at more distant sites. Moreover, two metazoan parasites, S. furciger (Digenea) and Echinorhynchus gadi (Acanthocephala), in the digestive were more abundant in samples taken at farther locations and also from the reference sites. These results, based on a laboratory study, are in agreement with previous observations that winter flounder exposed to sediment at the site nearest to the outfall, where high concentrations of toxic contaminants persisted, was greater than in the fish from the other locations.

Long-term studies on the reproductive fitness of fish under controlled exposure conditions are necessary to address some of the controversy surrounding the field-based studies of pulp and paper effluent effects. This study undertook effluent exposures of 2+ age rainbow trout that were approximately halfway through gonadal growth. Trout were exposed to a mixed thermomechanical/bleached kraft effluent in 12,000-L flow-through exposure tanks at an environmental research facility located at a pulp and paper mill in Kawerau, New Zealand. Trout were exposed to either upstream river water or 10% effluent in upstream river water and were maintained at a ration of 0.7% of body wet weight during the experiment. Results of the 2-month study indicated that trout survival was not significantly different between effluent-exposed tanks and reference tanks. There was extensive growth during the exposure but no differences were found due to effluent exposure. Gonadal development was not significantly different between treatments. Steroid hormone concentrations in males and females were not affected by effluent exposure. The effluent showed no potential to be estrogenic as indicated by a lack of vitellogenin induction in male trout. Other physiological indicators of energy storage and utilization also showed no significant differences. Modest induction of hepatic 7-ethoxyresorufin-O-deethylase (2.5-fold) was the only detectable biological effect of the exposure. Biliary concentration of effluent-related compounds were typical of pulp mill effluent exposure and further suggested that the source of phytosterols was in fact dietary and not effluent-derived.

As part of a larger survey on cumulative effects within the Saint John River basin (Canada), a fish survey was conducted near Edmundston (NB, Canada) in the fall of 1999 using slimy sculpin (Cottus cognatus) and white sucker (Catostomus commersoni). The discharge environment receives effluent from the pulp mill, a paper mill, three sewage discharges, and tributaries receiving agricultural runoff. Sculpin collected downstream of the sewage discharges and pulp mill effluent had greater growth, condition, and liver size but no significant differences in gonad size. Stable isotope data indicated slimy sculpin did not move between sites. Female sculpin collected downstream of the paper mill showed no significant differences in length, body weight, age, condition factor, liver size, and gonad size compared to fish from reference sites. Female white sucker collected downstream of the pulp mill did not differ significantly in any measured parameter compared to reference fish. Liver sizes of white sucker from the Saint John River were outside the range considered to be indicative of uncontaminated riverine sites. In 2000, sculpin collected downstream from a poultry-processing facility had larger livers and lower condition factors, suggesting that the site is contaminated. We found no significant differences in sculpin length, weight, condition (except for males), and liver size in sculpin collected downstream from the pulp mill in October 2001. The responses of slimy sculpin and white sucker differed, perhaps in relation to differences in life history characteristics. Results from this study indicate the slimy sculpin is a suitable fish species for monitoring rivers that receive multiple industrial and municipal effluents.

A study was conducted to determine the influence of decontamination on winter flounder living downstream from a pulp and paper mill. The fish and controls were held 8-26 weeks under contaminant-free conditions in aquaria supplied with ambient sea water. Groups of flounder, for comparison, were also sampled near the paper mill at the time of or following autopsy. No differences were apparent in length/organ-weight relationships between depurated and control groups after 26 weeks at 0-6 degrees C, but condition factor, gonadal and hepatosomatic indices, coincident with elevated levels of detoxifying enzymes, differed from field-derived samples. Pathological changes, including hemosiderin deposits in the liver and spleen, decreased, whereas pericholangitis and clear cell foci were apparent in the depurated fish and in the field samples. In two additional groups decontaminated for 8-16 weeks at 6-16 degrees C, there was no evidence of pericholangitis, but hemosiderin deposits persisted and an increase of fibrosis and clear cell foci occurred in contrast to the field group. These results suggest that some tissue repair in the liver transpired primarily during summer while other lesions, such as preneoplastic clear cell foci, which were probably induced prior to depuration, increased, but hemosiderin concentration remained unchanged. The latter observation is consistent with previous studies that suggest that hemosiderin concentration is not seasonally but age influenced.

This study assessed the effects of discharges from a sulphite-bleaching paper mill on winter flounder (Pleuronectes americanus) sampled each spring over a 5-year period in St. George’s Bay, Newfoundland, prior to foraging activity. The fish were captured by SCUBA divers near the mill and at a reference site 10 km up current. Several bioindicators were used to assess fish health. Larger and older flounder exhibiting gross and microscopic tissue lesions, lower condition factor, and elevated hepatosomatic index but lower gonadosomatic index that was associated with delayed development, were observed in samples caught near the mill compared to those at the reference site. Additionally, fish examined near the mill were infested with an ectoparasite, Cryptocotyle lingua, that causes black spot disease but harbored fewer numbers of a digene, Steringophorus furciger, in the digestive tract than samples taken at the reference site. These results suggest that abnormal size distribution, interruption of growth, high prevalence of lesions, lower condition factor, enlarged liver, delayed gonadal development, and differences in parasitic levels were indicative of stress in winter flounder caused by discharges from the mill compared to samples from a reference site. There was no evidence of a population decline in the inlet because of annual recruitment possibly from St. George’s Bay into which it opens.

Rainbow trout were exposed to a secondary treated, thermomechanical/bleached kraft pulp and paper effluent in 12,000-L, flow-through exposure tanks at an environmental research facility located at a pulp and paper mill in Kawerau, New Zealand. Trout (age, 2+ years) were obtained from a local hatchery and exposed either to upstream river water or a nominal concentration of 12% (v/v) effluent diluted in upstream river water. Three treatment groups were used: Effluent exposure that started approximately three months before gonadal growth (eight-month total exposure), effluent exposure that started approximately halfway through gonadal development (two-month total exposure), and trout exposed to reference water alone for the total duration of the experiment. Trout were sacrificed just before spawning; exposure, growth, and reproductive endpoints were assessed during and at the termination of the experiment. Reduction in growth was observed in both sexes in the eight-month treatment group relative to the river water reference treatment group. No differences were observed in condition factor or liver size in either treatment. Females in the eight-month exposure group also had significantly lower ovary weight. The two-month exposure group showed no differences from the reference group in growth or somatic indices. Estradiol and testosterone were reduced in blood samples taken from the eight-month exposure group by four months into the experiment as compared to the reference treatment. Steroid and vitellogenin levels in individual female trout from this treatment were significantly correlated with gonadosomatic indices (GSI) measured at the termination of the experiment. The GSI was not correlated strongly or consistently with pregnenolone, nor were any treatment-related pregnenolone differences observed, indicating that the steroid hormone reductions likely were not related to cholesterol side-chain cleavage. Male trout showed significant induction of vitellogenin and lower 11-ketotestosterone during the experiment (only the eight-month group was examined), but this did not result in any significant differences in testes development. Thus, this study has shown an impact of pulp mill effluent exposure on the reproductive physiology of female trout that appeared to be hormonally mediated. Furthermore, the effect could only be manifest when the exposure was initiated before the start of gonad development.

EROD (ethoxyresorufin-O-deethylase) activity was determined in winter flounder, a sediment-inhabiting and non-migratory fish species, living near a pulp and paper mill in Newfoundland in relation to temperature, gender, sexual maturity, and lesions in the liver. Samples of liver were taken from fish captured by SCUBA divers at 0 degrees C, 5 degrees C and 16 degrees C. Enzymic activity was detected in fish living only above 0 degrees C. Adult males and juvenile fish had higher levels of EROD activity than prespawning females at 5 degrees C. Macrophage aggregates only or occurring simultaneously with bile ductule hyperplasia and clear cell foci in the liver, did not impair EROD activity but necrosis had a negative effect. Results from this study indicate the importance of water temperature, gender, sexual maturity and liver pathology in assessing EROD activity of fish in biomonitoring programs.

Item does not contain fulltext We recommend that intestinal failure associated liver disease (IFALD) should be diagnosed by the presence of abnormal liver function tests and/or evidence of radiological and/or histological liver abnormalities occurring in an individual with IF, in the absence of another primary parenchymal liver pathology (e.g. viral or autoimmune hepatitis), other hepatotoxic factors (e.g. alcohol/medication) or biliary obstruction. The presence or absence of sepsis should be noted, along with the duration of PN administration. Abnormal liver histology is not mandatory for a diagnosis of IFALD and the decision to perform a liver biopsy should be made on a case-by-case basis, but should be particularly considered in those with a persistent abnormal conjugated bilirubin in the absence of intra or extra-hepatic cholestasis on radiological imaging and/or persistent or worsening hyperbilirubinaemia despite resolution of any underlying sepsis and/or any clinical or radiological features of chronic liver disease. Nutritional approaches aimed at minimising PN overfeeding and optimising oral/enteral nutrition should be instituted to prevent and/or manage IFALD. We further recommend that the lipid administered is limited to less than 1 g/kg/day, and the prescribed omega-6/omega-3 PUFA ratio is reduced wherever possible. For patients with any evidence of progressive hepatic fibrosis or overt liver failure, combined intestinal and liver transplantation should be considered.

The accumulation of ligands for the aryl hydrocarbon receptor (AhR) and fish sex steroid receptors was investigated using two separate controlled fish exposures to final effluent from a bleached sulfite/groundwood mill in New Brunswick, Canada. In the first experiment, hepatic tissue extracts from exposed fish were fractionated according to lipophilicity. Fractions with different octanol-water (Kow) partition coefficients were tested for the presence of bioavailable chemicals that function as ligands for the AhR in H4IIE cells, rainbow trout hepatic estrogen receptors (ER), goldfish testicular androgen receptors (AR), and goldfish sex steroid binding protein (SSBP). Fish accumulated ligands for each receptor after 4-d exposure to effluent. Single fractions contained ligands for the AhR and the ER, while multiple fractions competed for the AR and SSBP. Fish also accumulated ligands for the AhR and SSBP from Saint John River dilution water, indicating upstream sources of bioactive substances. Semipermeable-membrane devices deployed concurrently with fish accumulated ligands from effluent for all receptors except the ER. In the second experiment, accumulated ligands were evaluated after exposure of fish to effluent for two different durations and following a depuration period. Hepatic mixed function oxygenase activity and whole-liver hormonal activity, measured as binding to SSBP, returned to background following 6 d depuration and were reduced but still significant after 12-d exposure to effluent. Whole-liver extract affinities for the AR were maintained after extended exposure and depuration, indicating the potential for AR ligands to bioaccumulate. The accumulation of AhR ligands and ligands for sex steroid receptors provides a mechanistic linkage to effects on growth, development, and performance of fish exposed to effluent from this and other mills.

The study reported in this paper tested the hypothesis that the developmental and reproductive health of mosquitofish (Gambusia affinis) exposed to pulp and paper effluent in the Dengcun River would differ from that of mosquitofish living in a reference site. We also studied whether morphological characteristics such as the anal fin and hemal spines of mosquitofish could serve as indicators for evaluating the androgenic effect and mosquitofish population security in the Dengcun River. Male and female mosquitofish were captured at three sites contaminated by pulp and paper effluent in the Dengcun River in Sihui, South China, and at a nearby uncontaminated reference site. Samples were collected from the sampling sites on the same day in August 2009. We compared the populations by total length, wet body and liver mass, gonad mass, and population composition. We also compared the populations according to number of anal fin segments, oocyte and embryo count, anal fin and hemal spine morphology among females, and by sperm count and viability among males, and observed the gonadal and liver histology of both males and females. Female mosquitofish exposed to pulp and paper effluent in the Dengcun River were generally smaller in length and mass, had a greater number of anal fin segments and more embryos, but had significantly fewer oocytes in comparison with those living at the reference site. The higher number of anal fin ray 3 segments and the increased ray 4:6 length ratio observed among fish taken from the Dengcun River sites indicated that they might be subject to the androgenic effect. Furthermore, the significantly different hemal spine morphology of the effluent-affected females also indicated the pulp and paper mills effluents in Dengcun River might contain androgenic substance(s). Male mosquitofish at the sites exposed to effluent had a higher number of anal fin segments and greater testis mass in comparison with those living at the reference site. No evidence of intersex was found in either males or females, although histopathological tests on females revealed histologic abnormalities in the liver and gonads. It can be concluded that pulp and paper effluent contamination in the Dengcun River has affected a number of developmental parameters and reproductive characteristics in mosquitofish, with possible adverse effects on reproduction in this population.

Enzymatic and non-enzymatic antioxidants serve as an important biological defense against environmental oxidative stress. Information on antioxidant defense in fish is meager despite that fish are constantly exposed to a myriad of environmental stress including the oxidants. This study, therefore, assesses the activities of antioxidant enzymes viz., glutathione peroxidase, catalase and glutathione S-transferase and the non-enzymatic antioxidants viz., glutathione and metallothionein in various tissues of freshwater fish Channa punctatus (Bloch), in response to short-term and long-term exposures to paper mill effluent. The fish were exposed to the effluent at a concentration of 1.0% (v/v) for 15, 30, 60 and 90 days. The exposure caused a time-dependent increase in glutathione level (P0.001), activities of glutathione peroxidase (P0.05 to P0.001), glutathione S-transferase (P0.001) and a marginal initial decrease in catalase activity in the liver (P0.01 to P0.001). Metallothionein was induced in liver after 60 days of exposure. Two isoforms of metallothionein were detected. Catalase activity also increased 60 days afterwards. Antioxidant pattern was different in gill and kidney showing that liver was more resistant to oxidative damage as compared to gills and kidney. Our results demonstrate a pollutant-induced adaptive response in fish. In addition, levels of enzymatic and non-enzymatic tissue antioxidants may serve as surrogate markers of exposure to oxidant pollutants in fish.

Several biomarkers are currently used to determine chronic exposure of fish to environmental contaminants. In studies to assess the effects of pulp mill effluent on winter flounder (Pleuronectes americanus), body and hepatosomatic indices (HSIs), histopathology, levels of hepatic detoxifying enzymes, mixed function oxygenases (MFO), muscle acetylcholinesterase (Ache) and selected parasite abundance were compared in samples taken near a paper mill (sulphite-bleaching) and a number of reference sites. Our studies revealed a number of significant (P0.05) differences between samples taken near the paper mill and reference sites. Flounder captured at two locations near the mill were greater in length and weight, had elevated HSIs and more lesions in the liver and spleen while an acanthocephalan, Echinorhynchus gadi, located in the digestive tract, was lower than at more distant sites. Activity of MFO and Ache were similar at most sites and might be a reflection of the fish's foraging behaviour and consequently intermingling of populations from different locations during summer. These results support the view that several biomarkers should be utilised when assessing the health of fish living in habitats degraded by pollutants.