Temporal stability and rates of post-depositional change in geochemical signatures of brown trout Salmo trutta scales.

This study investigates temporal stability in the scale microchemistry of brown trout Salmo trutta in feeder streams of a large heterogeneous lake catchment and rates of change after migration into the lake. Laser-ablation inductively coupled plasma mass spectrometry was used to quantify the elemental concentrations of Na, Mg, Mn, Cu, Zn, Ba and Sr in archived (1997-2002) scales of juvenile S. trutta collected from six major feeder streams of Lough Mask, County Mayo, Ireland. Water-element Ca ratios within these streams were determined for the fish sampling period and for a later period (2013-2015). Salmo trutta scale Sr and Ba concentrations were significantly (P < 0·05) correlated with stream water sample Sr:Ca and Ba:Ca ratios respectively from both periods, indicating multi-annual stability in scale and water-elemental signatures. Discriminant analysis of scale chemistries correctly classified 91% of sampled juvenile S. trutta to their stream of origin using a cross-validated classification model. This model was used to test whether assumed post-depositional change in scale element concentrations reduced correct natal stream classification of S. trutta in successive years after migration into Lough Mask. Fish residing in the lake for 1-3 years could be reliably classified to their most likely natal stream, but the probability of correct classification diminished strongly with longer lake residence. Use of scale chemistry to identify natal streams of lake S. trutta should focus on recent migrants, but may not require contemporary water chemistry data.

Thermal, trophic and metabolic life histories of inaccessible fishes revealed from stable-isotope analyses: a case study using orange roughy Hoplostethus atlanticus.

A time-resolved record of inhabited water depth, metabolic rate and trophic behaviour of the orange roughy Hoplostethus atlanticus was recovered from combined stable-isotope analyses of otolith and muscle tissue. The results demonstrate that H. atlanticus from the north-east Atlantic Ocean have a complex life history with three distinct depth-stratified life stages. Early juvenile H. atlanticus occupy relatively shallow habitats, juvenile H. atlanticus show a deep-demersal phase, rising at sexual maturity, and adult H. atlanticus exploit increasingly deep habitats with increasing age. At all sampled sizes, H. atlanticus muscle tissues have an isotopic composition suggesting a benthic rather than benthopelagic or pelagic diet. Isotopic measures of relative metabolic rate provide an insight into energy partitioning throughout ontogeny. Hoplostethus atlanticus have relatively low metabolic rates compared to coexisting deep-water benthic fishes, consistent with their unusually high longevity. Surprisingly, lifetime fastest growth rates are achieved during juvenile stages when otolith isotopes imply deep-water residency and relatively low metabolic rates. Fast growth may be sustained during a period of high efficiency associated with reduced metabolic costs of prey capture or predator evasion. The stable-isotope approach can be applied to any teleost and provides a rapid, cost-effective technique for studying deep-water fish communities.

Density-independent growth of floodplain river channel catfish Ictalurus punctatus.

This study investigated the relative influence of biotic and abiotic factors on the growth of channel catfish Ictalurus punctatus in seven Mississippi floodplain rivers. The results indicate that growth was density-independent, being defined largely by abiotic conditions.

Pharmacokinetic behaviour of sublingually administered 8-methoxypsoralen for PUVA therapy.

BACKGROUND: Conventional oral PUVA therapy is hampered by large inter- and intraindividual variations in the bioavailability of 8-methoxypsoralen (8-MOP), caused by its low solubility in the gastrointestinal juices and large interindividual differences in hepatic metabolism rate (hepatic first pass). AIMS: New galenic formulations of 8-MOP based on solid dispersions, suspensions, and saturated solutions containing penetration enhancers were developed for sublingual administration, a drug delivery route which avoids the hepatic first pass metabolism. METHODS: Solubility properties of 8-MOP were tested in 22 potential penetration enhancers and solubilizers. Following preliminary in vivo tests of 13 sublingual 8-MOP formulations, five were administered to groups of volunteers at a nominal dose of 0.6 mg/kg body weight: two solid dispersions based on PEG 1540 (with and without Xylitol); a solution in Labrasol (glyceryl and PEG-8 caprylate/caprate), PEG 400, Transcutol (ethoxydiglycol) (1:1:1); a micronized suspension in sorbitol, water, ethanol, propylene glycol (ca. 3:1:1:5 w/v); and Oxsoralen capsules. Pharmacokinetic behaviour of 8-MOP was examined in serum; samples were analysed by HPLC. Photosensitivity was measured in seven subjects. RESULTS: The peak of maximum 8-MOP concentration in blood was sharp, rapid and reproducible: tmax of 8-MOP in blood averaged 23+/-3 min, independent of the particular formulation. Cmax was higher when 8-MOP was presented in dissolved form (solution and capsule formulations, 85+/-29 and 85+/-35 ng/ml, respectively) and lowest with the suspension (42+/-15 ng/ml). Photosensitivity peaked reproducibly at 45 min. post dosing. CONCLUSIONS: Sublingual PUVA therapy is suitable for patients with skin types I and II, in particular patients who are less suitable candidates for standard PUVA therapy (due to hepatic, renal, or cardiac insufficiency) or who have experienced side effects with standard PUVA.

Carcinogenic risk of bath PUVA in comparison to oral PUVA therapy.

The potential carcinogenic risk of bath PUVA therapy was compared to that of systemic (oral) PUVA. An analysis of the epidemiological data on cancer risk following bath PUVA with trimethylpsoralen does not support the conclusion that bath PUVA per se is less carcinogenic than systemic PUVA with 8-methoxypsoralen (8-MOP). Pharmacokinetic studies indicate that both the concentration of 8-MOP in the target organ for PUVA carcinogenicity (skin) at the relevant time point (time point of UVA irradiation) and the extents of biological effects in the skin are comparable following bathwater or systemic 8-MOP administration. Furthermore, the therapeutic effects of PUVA arise from the same photochemical reaction mechanisms as do the carcinogenic effects. Theoretically, the ratio of (desired) cytotoxic versus (undesired) mutagenic effects could increase with increasing efficiency of the PUVA therapy itself. On the basis of the available evidence, it is concluded that all forms of PUVA therapy, independently of the route of 8-MOP administration, contribute to a small but dose-dependent increase in nonmelanoma skin cancer risk.

Measurement of 5-methoxypsoralen and 8-methoxypsoralen in saliva of PUVA patients as a noninvasive, clinically relevant alternative to monitoring in blood.

BACKGROUND: Monitoring of psoralen concentration and time-course in PUVA patients is vital for efficient PUVA therapy. Blood sampling is invasive and labour-intensive and thus unsuited for routine use and repeat measurements over the course of therapy. OBJECTIVE: Psoralen pharmacokinetics in saliva were investigated and validated as a noninvasive, simple and biologically relevant alternative to measurements in blood. METHODS: The time-course of psoralen concentration was measured in saliva and serum of volunteers and patients receiving PUVA or extracorporeal photopheresis therapy. The samples were analysed by high-performance liquid chromatography. Three commonly used oral psoralen preparations were tested: Psoraderm5 (5-methoxypsoralen; 5-MOP), Meladinine and Oxsoralen (both 8-methoxypsoralen; 8-MOP). RESULTS: The pharmacokinetic parameter Cmax in saliva averaged 10% (range 6-20%) of the serum values for 8-MOP, and < or = 4% for 5-MOP. These concentrations correspond to the therapeutically relevant, non-albumin-bound fraction of psoralen in serum that is available to diffuse into the tissues. The parameter tmax in saliva and serum coincided, indicating that psoralens diffuse rapidly between the two compartments. CONCLUSION: Monitoring of psoralens in saliva is a valuable, noninvasive alternative to measurements in serum, suitable for routine use. A series of five or six saliva samples is sufficient to determine tmax in a patient beginning photochemotherapy. To determine Cmax, three independent saliva measurements at t = tmax are recommended.

Autotransfusion of washed shed mediastinal fluid decreases the requirement for autologous blood transfusion following cardiac surgery: a prospective randomized trial.

OBJECTIVES: The National Blood Service issues 2.2 million units of blood per year, 10% of these (220000) are utilized in cardiac procedures. Transfusion reactions, infection risk and cost should stimulate us to decrease this transfusion rate. We test the efficacy of autotransfusion following surgery in a prospective randomized trial. METHODS: One hundred and twelve patients undergoing CABG, valve or CABG + valve procedures were randomized into two groups. Group A received washed postoperative drainage fluid and group C were controls. The indication for transfusion was a postoperative haemoglobin (Hb) < 10 g/l or a PCV < 30. There was no significant difference in preoperative and operative variables between the groups. RESULTS: Twenty-eight patients in group A and 46 in group C required homologous transfusion (P = 0.0008). Group A patients required 298+/-49 ml of banked blood per patient, group C 508+/-49 ml (P = 0.003). There was no difference in total blood required (volume autotransfused + volume banked blood transfused) between the groups (group A 404+/-50 ml, group C 508+/-50 ml) or in mean total mediastinal fluid drainage (group A 652+/-51 ml, group C 686+/-50ml). The mean Hb concentration was significantly higher in group A on day 1 (11.2 g/dl+/-51 vs. 10.6 g/dl+/-13 (P = 0.002)). No morbidity was associated with autotransfusion. CONCLUSION: Autotransfusion can decrease the amount of homologous blood transfused following cardiac surgery. This represents a benefit to the patient and a decrease in cost to the health service.

Pharmacokinetics of 8-methoxypsoralen during extracorporeal photopheresis.

BACKGROUND/PURPOSE: Extracorporeal photopheresis (ECP) is a widely used therapy for the treatment of diverse diseases such as cutaneous lymphomas and graft-vs-host disease. Knowledge of the effective concentration of 8-methoxypsoralen (8-MOP) in the photopheresis apparatus and the photodegradation time-course of 8-MOP during ECP is a prerequisite for a successful therapy. METHODS: The time course of 8-MOP concentration was measured in patients' serum and in the photoactivation chamber (so-called buffy coat fraction) during ECP. Samples were analyzed by high-performance liquid chromatography. Half-lives of 8-MOP in both fractions were calculated assuming first-order kinetics (exponential decay). Losses due to adsorption and photodegradation were investigated and the recovery of bioavailable 8-MOP calculated. RESULTS: In female patients (average age 61+/-9 years) given 0.4-0.6 mg 8-MOP/kg body weight in the form of Oxsoralen capsules, peak serum concentrations averaged 420+/-80 ng/ml (n=8). In contrast, peak concentrations in the photoactivation chamber averaged only 134 ng/ml, or 32% of serum values. In serum, peak 8-MOP concentrations were reached < or =40 min following ingestion; the half-life of 8-MOP in the serum was 50+/-14 min (n=7). The effective half-life of 8-MOP in the photoactivation chamber was considerably longer (about 4 h). The recovery of free, bioavailable 8-MOP in the photoactivation chamber at the end of ECP averaged 42% of the applied dose; losses stemmed mainly from photodegradation of 8-MOP and from adsorption of 8-MOP to the surfaces of the apparatus. CONCLUSION: We conclude that interpretation of investigations on clinical success and dose-response aspects of ECP must take into account the complex pharmacokinetic behaviour of 8-MOP during the ECP procedure.

Covalent binding of agaritine to DNA in vivo.

14C-Ring-labelled agaritine was administered orally to eight C57BL/6 mice at a chemical dose of 7.5 mg and radioactive dose of 1.2 x 10(9) dpm/kg body weight. After 24 hr, the animals were killed and DNA from stomach, liver and kidneys was purified by a phenol-free method involving proteinase K digestion of chromatin and coprecipitated proteins, followed by hydroxylapatite chromatography, dialysis and precipitation with ethanol. An increase in radioactivity was found in DNA of all three organs examined. Stomach DNA had the highest levels: 160 and 30 dpm/mg DNA in males and females, respectively. Liver and kidney DNA both showed levels of approximately 1 dpm/mg, with no measurable gender differences. Expressed in the units of the covalent binding index (CBI), agaritine has a potency of 42 in mouse stomach in males and 8 in females. The CBI of agaritine in liver and kidney was 0.2-0.3 in both sexes. The genotoxic activity of agaritine is thus very weak. The cumulative lifetime cancer risk of agaritine consumption in mushrooms is estimated to lie at approximately 10(-5).

Comparison of 8-MOP aqueous bath and 8-MOP ethanolic lotion (Meladinine) in local PUVA therapy.

BACKGROUND AND OBJECTIVE: Local PUVA (psoralen plus UVA light) is an effective outpatient treatment for patients with palmoplantar eczema or psoriasis. In this study, the efficacy, applicability and patient acceptance of two local forms of 8-methoxypsoralen (8-MOP) PUVA therapy were compared. METHODS: The study design was a left-right comparison (n = 37): the left hand or foot was treated with (aqueous) 8-MOP bath PUVA whereas the right received (ethanolic) 8-MOP lotion PUVA. After 1 month, the more successful treatment was continued on both sides until lesions cleared. RESULTS: Both therapies were effective and both useful for particular clinical applications: patients with erosive lesions and rhagades appreciated the gentleness of bath PUVA. Those with pustules or hyperkeratotic lesions appreciated the greater effectiveness of 8-MOP lotion PUVA. The total UVA dose and number of sessions to clearance were smaller with 8-MOP lotion. There was no difference in the length of the relapse-free period. Therapy nonresponders usually became apparent within the first 12 sessions. CONCLUSIONS: The difference between bath PUVA and lotion PUVA can be described as 'gentle' versus 'strong' therapy. The better therapy depends on clinical indication.

Statistical analysis of the lacI transgenic mouse mutagenicity assay.

The transgenic mouse assay is now widely used to test chemicals for genotoxic potential. In this article, we consider statistical tests for increasing trend in mutant frequency with increasing dose, along with statistical models that may be used to describe the observed dose-response relationships. The application of these methods is illustrated using data on 2-acetylaminofluorene, di(2-ethylhexyl)phthalate, heptachlor, and sodium phenobarbital. No strong evidence of extra-binomial variation was detected at the plate level, but greater evidence was noted when the data were aggregated to the package or animal level in liver, necessitating the use of statistical methods that allow for overdispersion relative to binomial variation. Clear increase on mutant frequency induced by 2-acetylaminofluorene was detected in both liver and bladder, but no apparent trends were noted with di(2-ethylhexyl)phthalate, heptachlor, and sodium phenobarbital. The exponential model provides a good fit to the observed dose-response relationship in liver, whereas a Weibull model provides a better fit for bladder.

Genotoxicity of agaritine in the lacI transgenic mouse mutation assay: evaluation of the health risk of mushroom consumption.

The mutagenic potency of the common mushroom Agaricus bisporus and crude agaritine extracted from mushrooms was determined in vivo using a new mutagenesis assay with lacI transgenic mice (Big Blue mice). Pairs of female lacI mice were fed one of three diets for 15 wk: (1) fresh mushrooms 3 days/wk followed by normal lab chow for 4 days/wk; (2) freeze-dried mushrooms mixed at 25% (w/w) into powdered chow; or (3) a mushroom extract containing 30% agaritine (w/w) mixed into powdered chow. The corresponding daily doses of agaritine were 30 (averaged over the whole week), 80 and 120 mg/kg body weight, respectively. Positive control animals received N-nitrosodimethylamine, N-nitrosomethylurea or urethane, mixed into powdered chow at concentrations corresponding to daily doses of 0.3, 3 and 130 mg/kg body weight, respectively. DNA of the forestomach, kidney, liver, lung and glandular stomach of the lacI mice was examined for increases in mutant frequency (MF). Control MFs ranged from 5 x 10(-5) to 10 x 10(-5). Positive control substances induced a two- to seven-fold increase in MF in their respective target organs. Of the mushroom diets, significant effects were seen only with the crude agaritine extract: it induced an increase in MF of 100% in the kidney and 50% in the forestomach. The other two A. bisporus diets, with lower agaritine doses, showed slightly but not significantly, raised MF values in the kidney alone. Thus, agaritine was weakly genotoxic in vivo; no genotoxic activity other than that attributable to agaritine was detected in A. bisporus. Substances or processes that might influence carcinogenicity by means of non-genotoxic mechanisms (e.g. increase in fibre, or decrease in calorie intake) are not detected in the lacI assay. Using a previously derived quantitative correlation between mutagenicity in the lacI test and carcinogenic potency, the carcinogenicity of agaritine in mushrooms was estimated: the average Swiss mushroom consumption of 4 g/day would be expected to contribute a lifetime cumulative cancer risk of about two cases per 100,000 lives.

The lacI transgenic mouse mutagenicity assay: quantitative evaluation in comparison to tests for carcinogenicity and cytogenetic damage in vivo.

The detection limit of the lacI transgenic mouse mutagenicity assay lies, in practice, at approximately a 50-100% increase in mutant frequency in treated animals over controls. The sensitivity of this assay in detecting genotoxins can be markedly improved by subchronic rather than acute application of the test compound. The lacI transgenic mouse mutagenicity assay was compared quantitatively to rodent carcinogenicity tests and to presently used in vivo mutagenicity assays. With the genotoxic carcinogens tested thus far, a rough correlation between mutagenic potency and carcinogenic potency was observed: on average, to obtain a doubling in lacI mutant frequency the mice had to be treated with a total dose equal to 50 times the TD50 daily dose level. This total dose could be administered either at a high dose rate within a few days or, preferably, at a low dose rate over several weeks. This analysis also indicated that a lacI experiment using a 250-day exposure period would give a detection limit approximately equal to that of a long-term carcinogenicity study. In comparison to the micronucleus test or the chromosome aberration assay, acute studies with the presently available lacI system offered no increase in sensitivity. However, subchronic lacI studies (3-4-month exposure) resulted in an increase in sensitivity over the established tests by 1-2 orders of magnitude (shown with 2-acetylaminofluorene, N-nitrosomethylamine, N-nitrosomethylurea and urethane). It is concluded that a positive result in the lacI test can be highly predictive of carcinogenicity but that a negative result does not provide a large margin of safety.

Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene.

2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue mice). The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10(5) plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10(-5) (pooled results of two animals, 8 mutant plaques/289,530 plaques). At 300 ppm in the diet, the rate of 3.5 x 10(-5) (8/236,300) was not significantly increased over background. At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 x 10(-5) (17/221,240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF.

Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation.

Naturally occurring dipeptides, cholecystokinine (CCK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10-30 min with 40 mM-nitrite (pH 3.5, 37 degrees C), and the resultant products examined for mutagenicity in Salmonella typhimurium TA100. Specific mutagenicities (net revertants per mumol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/mumol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/mumol). Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/mumol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/mumol, respectively). The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/mumol, respectively). Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group. The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains. The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation. Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach. Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.

The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).

Effect of very low calorie diet on body composition and exercise response in sedentary women.

The effect of very low calorie diet (VLCD) on fat-free mass (FFM) and physiological response to exercise is a topic of current interest. Ten moderately obese women (aged 23-57 years) received VLCD (1695 kJ.day-1) for 6 weeks. FFM, estimated by four conventional techniques, and heart rate (fc), blood lactate (la(b)), mean arterial pressure (MAP), respiratory exchange ratio (R) and rating of perceived exertion (RPE) were measured during a submaximal cycle ergometry test 1 week before, in the 2nd and 6th week, and 1 week after VLCD treatment. Strength and muscular endurance of the quadriceps and hamstrings were tested by isokinetic dynamometry. The 11.5-kg reduction in body mass was approximately 63% fat and 37% FFM. The latter was attributed largely to the loss of water associated with glycogen. Whilst exercise fc increased by 9-14 beats.min-1 (P < 0.01), there were substantial decreases (P < 0.01) in submaximal MAP (1.07-1.73 kPa), la(b) (0.75-1.00 mmol.l-1 and R (0.07-0.09) during VLCD. R and fc returned to normal levels after VLCD. Gross strength decreased (P < 0.01) by 9 and 13% at 1.05 rad.s-1 and 3.14 rad.s-1, respectively. Strength expressed relative to body mass (Nm.kg-1) increased (P < 0.01) at the lower contraction velocity, but there was no change at the faster velocity. Muscular endurance also decreased (P < 0.01) by 62 and 82% for the hamstrings and quadriceps, respectively. We concluded that the strength decrease was a natural adaptation to the reduction in body mass as the ratio of strength to FFM was maintained.(ABSTRACT TRUNCATED AT 250 WORDS)