Transport and acclimation conditions for the use of an estuarine fish (Pomatoschistus microps) in ecotoxicity bioassays: effects on enzymatic biomarkers.

Acclimation of organisms for ecotoxicity testing is in general processed according to Organisation for Economic Co-operation and Development (OECD) and/or Environmental Protection Agency (EPA) guidelines, under controlled conditions. However, when organisms are collected in the field, their capture, transport and adaptation to laboratory conditions are factors of stress. In their natural habitat, estuarine fish are exposed to considerable fluctuations of environmental variables, while in laboratory they are acclimated to constant conditions and this can be per se a factor of stress that may influence biomarker responses. Therefore, it is important to investigate the effects of these procedures on estuarine fish performance before using them as test organisms in ecotoxicity bioassays. The goal of the present study was to investigate the effects of transporting the common goby, Pomatoschistus microps from the field (natural populations) to the laboratory and of its acclimation to laboratory conditions on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GSTs). Fish were collected in a reference site of the Minho River estuary (NW of Portugal) and the activities of the biomarkers were monitored before and after the transport of organisms to the laboratory and during the acclimation period (at 5, 10 and 15 days). The activities of all the enzymes indicated that capture and transport conditions had no effects on enzymatic activities. Furthermore, AChE, LDH and GST presented higher activities at the end of the acclimation period than at beginning, suggesting a physiological adaptation to laboratory conditions. This adaptation should be taken into consideration in the experimental design to avoid bias in the interpretation of effects of xenobiotics on biomarkers.

Estimation of postmortem interval. Temperature-correction of extracellular abdominal impedance during the first 21 days of death.

Extracellular impedance of the intact abdomen of rats increased from 79.0+/-7.4Omega, 1h postmortem (i.e. 0.04 day), to 130.5+/-14.4Omega at postmortem interval (PMI)=1 day. Impedance then decreased with time, reaching 64.2+/-9.9Omega at PMI=21 days. The time taken for mean abdominal impedance to decrease below the value at PMI=0.04 day averaged 16 days. It is therefore impossible, using extracellular abdominal impedance alone, to distinguish (in terms of interpolating PMI) between numerically equal impedances on the rising and falling phases of curves depicting impedance as a function of PMI. Correction of measured impedances to their theoretically-predicted values at an arbitrarily chosen temperature of 40 degrees C appreciably diminished the magnitude of the increase in impedance following death. Thus, temperature-corrected abdominal impedance increased from 56.2+/-4.8Omega at PMI=0.04 day to 59.5+/-6.2Omega at PMI=1 day. Impedance then decreased, reaching 29.2+/-4.1Omega at PMI=21 days. The time taken for mean, temperature-corrected abdominal impedance to decrease below the value at PMI=0.04 day averaged 3 days (as opposed to 16 days (see above) in the absence of temperature-correction). These findings are believed to improve the usefulness of extracellular abdominal impedance as a potential tool for estimation of postmortem interval.

Temperature-correction of abdominal impedance: improved relationship between impedance and postmortem interval.

The relationship between extracellular abdominal impedance and postmortem interval (PMI) reflects the combined effects, on impedance, of postmortem cooling of the tissues and of autolysis per se. This study was performed in order to eliminate temperature change as a major factor contributing to the time course of postmortem change in abdominal impedance. Dissociation of thermal and autolytic influences was achieved by recording deep abdominal temperature at the time of impedance measurement, followed by correction of all measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C. Uncorrected abdominal impedance increased from 82+/-12 Ohmz, 1 h after death, to 108+/-21 Ohmz after 12 h. Impedance then decreased to 96+/-23, 89+/-22, 75+/-19, 66+/-21 and 59+/-19 Ohmz at postmortem intervals of 24, 36, 48, 60 and 72 h, respectively. In contrast, corrected abdominal impedance decreased progressively from 63+/-7 Ohmz, 1 h after death, to 61+/-9, 56+/-11, 51+/-10, 46+/-10, 39+/-11 and 35+/-10 Ohmz at postmortem intervals of 12, 24, 36, 48, 60 and 72 h, respectively. The improved relationship between (corrected) abdominal impedance and PMI is of potential value in estimating time since death.

A preliminary study of changes in scalp impedance during the early post-mortem period in rats.

An accurate and reproducible technique was used for estimation of scalp impedance (1 kHz) in each of eight rat cadavers, maintained at 9.0 +/- 0.7 degrees C, during the 1 to 144 hour post-mortem period. Scalp impedance increased non-linearly, and in a bimodal manner, between 24 and 144 hours post-mortem. The relationship between scalp impedance and post-mortem interval, and the degree of individual variation in the time course of impedance change, were such that scalp impedance is unlikely to be a suitable tool for estimation of time since death.

A preliminary investigation into postmortem changes in skinfold impedance during the early postmortem period in rats.

A highly accurate and reproducible technique was used to measure the electrical impedance (1 kHz) of a lateral abdominal skinfold, in each of eight rat cadavers stored at 9.0 +/- 0.7 degrees C, during the early postmortem period. Impedance increased exponentially between 1 and 120 h post mortem. Whether or not similar changes occur during the human postmortem period, and their potential value as a means of objective estimation of postmortem interval (PMI), remains to be established.

Changes in the resistive and reactive components of abdominal impedance during the 1-21 day post-mortem periods in rats.

The magnitudes of the resistive (R) and reactive (X) components of total abdominal impedance (Zt) at 10 kHz, and of transcellular impedance (Zi), cytoplasmic impedance (Ri) and cell membrane capacitance (C), were calculated from sequential measurements of Zt, phase angle (theta) and extracellular impedance (Ze), in each of eight rat cadavers maintained at 9.0 +/- 1.5 degrees C. Total impedance, theta, R, X, Ze and Ri increased during the first 3 days of death, while C decreased. Thereafter, each of these parameters decreased with increasing post-mortem interval. The data indicate that Zt was predominantly resistive, and that Zt became progressively more resistive, and less reactive, with increasing time since death. Transcellular impedance remained relatively constant during the first 9 days, prior to increasing to more than 160% of its initial (3 h) value at 21 days post-mortem. This change coincided temporally with, and was attributable to, a time-dependent decrease in cell membrane capacitance post-mortem. Further studies, using human post-mortem tissues or body segments, will determine whether or not time-related changes in one or more of the impedance parameters may be used to estimate post-mortem interval.

Transcellular and extracellular impedances of the intact abdomen in putrefying rat cadavers.

Total (Zt) and extracellular (Ze) abdominal impedances of putrefying rat cadavers, stored at 18.0 +/- 0.7 degrees C, were measured at 50 kHz and at 1 kHz, respectively, during the first 7 days of death. Transcellular impedance was calculated from the measured values of Zt and Ze. Total impedance and Ze increased during the first day, and then decreased with increasing postmortem interval, while Zi remained unchanged during the first 3 days before increasing more than 4-fold between 3 and 7 days postmortem. These changes are consistent with net gain of electrolyte by the extracellular fluids and with degeneration of cell membranes which occur postmortem. It is concluded that, during putrefaction, Ze is a potentially more useful index of postmortem interval than Zt or Zi, and that measurement of total abdominal impedance at 50 kHz, rather than at 10 kHz, markedly improves the precision and accuracy of impedance measurement.

Time-dependent changes in electrical resistance of the intact trunk, thorax and abdomen of rats during the first 21 days post mortem.

Electrical resistance of the intact trunk, thorax and abdomen was determined sequentially in each of eight rat cadavers maintained at constant temperature. All electrodes applied to each cadaver were left in situ for the duration of the 21-day postmortem period. Trunk, thoracic and abdominal resistances increased (on average) from 337 +/- 31, 174 +/- 27 and 163 +/- 22 omega 1 h after death, to peak values of 567 +/- 46, 333 +/- 36 and 254 +/- 33 omega at 2, 3 and 1 days post mortem, respectively. Thereafter, all resistances decreased progressively with increasing time since death, reaching values of 294 +/- 39 omega (trunk), 173 +/- 33 omega (thorax) and 121 +/- 19 omega (abdomen) at 21 days post mortem. These changes are believed to reflect progressive postmortem decreases in the resistivities of the body segments studied.

Time-dependent changes in electrical resistance of the intact abdomen during the 1-504 h postmortem period in rats.

Eight rat cadavers, at controlled ambient temperature, were each equipped with electrodes for passing current of constant intensity through the cadaver and for measurement of the potential difference between two points on the anterior abdominal skin. All electrodes were left in situ for the duration of the study, and abdominal resistance was determined at 1, 5 and 24 h post mortem, and at 24-h intervals between 24-504 h post mortem. Abdominal resistance increased during the first 5-24 h and then decreased progressively between 24-480 h (one cadaver) or 24-504 h (seven cadavers) post mortem. These findings are believed to reflect a postmortem decrease in abdominal resistivity per se.

Postmortem changes in resistivity of the anterior abdominal wall during the early postmortem period in rats.

An accurate and reproducible technique was employed for the determination of the resistivity of excised portions of the anterior abdominal wall of rats. Resistivity decreased linearly (r = -0.93; P < 0.001) from 1438 +/- 131 omega.cm, immediately following death, to 360 +/- 144 omega.cm at a postmortem interval of 36 h. These changes are believed to reflect the morphological and/or chemical changes which occur at the cellular level during the early postmortem period.

Postmortem changes in electrical resistance of the gastric wall during the early postmortem period in rats.

A simple and reliable technique was employed for measurement of (dc) electrical resistance of the gastric wall of rats. Tissue resistance decreased linearly (r = -0.87; P less than 0.001) from 66.1 +/- 12.1 kOhm/cm2 immediately following death to 23.3 +/- 3.5 kOhm/cm2 at a postmortem interval of 24 h. These changes are believed to reflect progressive postmortem changes in the physical dimensions of the intercellular space and/or changes in electrical conductivity of the intercellular/extracellular fluids.

In vitro loss of potassium from erythrocytes during the 0-108 h postmortem period in rats: relationship between potassium loss and postmortem interval.

A simple technique was employed for measurement of potassium loss (K(los)s) from normal and from postmortem rat erythrocytes during controlled exposure to physiologically isosmotic NaCl solution. Potassium loss from the cells decreased in a (non-linear) time-related manner during the 0-108 h postmortem period; expression of the data in the form of a double logarithmic plot (log K(loss) versus log postmortem interval (PMI) linearized the relationship between 18 and 108 h post mortem (r = -0.86; P less than 0.001 (n = 24)). Experimental data revealed that the observed postmortem changes in K(loss) were associated with and probably resulted from, the postmortem decrease in magnitude of the potassium concentration gradient across the erythrocyte membrane. Attention is drawn to the possibility of utilizing measurements of in vitro loss of potassium from erythrocytes as a means of estimating time elapsed since death.

Plasma volume and renal function during and after ultramarathon running.

Plasma volume (PV) and renal function were studied in eight subjects for 3 d prior to and 6 d after a 56 km footrace. Immediately following the race, PV, creatinine clearance, and urine flow were unchanged from pre-race values. Over the subsequent 3 d, PV increased due initially to a 17 g influx of serum albumin and an associated increase in plasma sodium content, which persisted throughout the study period. A reduction in urine sodium secretion occurred during the race day. Creatinine clearance increased after the race and remained elevated for 48 h. Increases serum enzyme activities, C-reactive protein concentration, serum uric acid content, and plasma creatinine concentration and production suggest muscle damage. We suggested the following. First, the persistent post-exercise plasma volume expansion is initiated by an influx of albumin into the intravascular space with an associated increase in plasma sodium content. A decrease in urine sodium excretion during the race day would contribute to the latter. Second, the interpretation of post-race changes in serum constituents must take account of changes in plasma volume. Third, there is an increase in creatinine clearance, indicating an increase in glomerular filtration rate, after both standard and ultramarathon running. This may be caused by the products of muscle cell damage although the physiologic mechanism for this is unclear.

Linearization of the relationship between postmortem plasma chloride concentration and postmortem interval in rats.

A potentiometric titration procedure was employed for measurement of plasma chloride concentrations during the 0-96 h postmortem period in rats. The data revealed antemortem absolute values and postmortem rate of decrease in plasma chloride concentration (PCl) which were almost identical to those in dogs and in man. Expression of the data in the form of a double logarithmic plot of PCl versus postmortem interval (PMI) yielded a linear relationship of high correlation (r = -0.97; P less than 0.001). Attention is drawn to the possibility of utilizing postmortem rate of change in plasma chloride concentration as a means of estimating time elapsed since death.

Double logarithmic, linear relationship between plasma sodium/potassium concentration ratio and postmortem interval during the 6-96-h postmortem period in rats.

Flame photometric determination of sodium and potassium concentrations in postmortem rat plasma (n = 40) revealed the existence of a linear and inverse relationship, of high correlation (r = -0.986; P less than 0.001), between the logarithm of the plasma sodium/potassium concentration ratio (PNa/PK) and the logarithm of postmortem interval (PMI) during the 6-96-h postmortem period. This relationship, which has not been reported previously in the forensic literature, offers a simple and potentially valuable means of estimating the PMI in man.

Linear rate of change in the product of erythrocyte water content and potassium concentration during the 0-120-hour postmortem period in the rat.

An accurate and reproducible technique was employed for measurement of water content (RBCH2O) and potassium concentration (RBCK) in rat erythrocytes post mortem. Coefficients of variation for determination of RBCH2O and RBCK, as estimated from the results of duplicate analyses (n = 36), were 1.21% and 1.17%, respectively. Erythrocyte water content and RBCK were directly and linearly related (r = +0.93 (P less than 0.001], while the product of RBCH2O and RBCK varied linearly and inversely (r = -0.89 (P less than 0.001] with postmortem interval (PMI) over the 0-120-h postmortem period. In addition, the standard deviations of the data points for (RBCH2O X RBCK) remained relatively constant and independent of PMI. Attention is drawn to the possibility of determining time elapsed since death from (sequential) measurements of (RBCH2O X RBCK) in individual cadavers.

Blood-volume expansion in the rat: natriuresis accompanied by a fall in filtration fraction.

1. We have attempted to confirm the existence of a natriuretic hormone released in response to acute expansion of blood volume. 2. Isolated kidneys, perfused with whole blood at constant pressure, were incorporated within an extracorporeal circulation in recipient rats. In six control experiments urine flow rate, renal blood flow, glomerular filtration rate, filtration fraction, and the fractional excretion of filtered sodium and water were measured for periods of up to 120-140 min thereafter. The same variables were measured in a further 12 experiments in which, after 63 +/- 11 min, the rats were volume expanded with equilibrated whole blood (15, 18 or 28 ml/kg body wt). 3. On average the controls revealed no change in any of the variables measured; volume expansion was followed by increased renal blood flow and fractional excretion of filtered sodium and water, while the filtration fraction fell. 4. In both the control and volume-expansion experiments, there were 12 instances in which the fractional excretion of filtered sodium increased; in 10 of these, including those experiments in which the natriuresis was most marked, there was a closely correlated fall in filtration fraction. 5. In all the experiments changes in the fractional excretion of filtered sodium and water varied in parallel. 6. We conclude that volume expansion (a) changes the concentration of some circulating vasoactive substance(s) and (b) results in natriuresis and diuresis consequent upon a fall in filtration fraction.

Lack of effect of blood-volume expansion on short-circuit current across an isolated toad skin incorporated in the circulation of a rat.

1. Evidence for the existence of 'natriuretic hormone' resides, in part, in the demonstration that blood volume expansion in the dog is followed by a transient fall in short-circuit current (SCC) across a frog skin incorporated within its circulation. 2. We have attempted to confirm this effect in the rat, with a toad skin (Xenopus laevis) incorporated within the circulation. The skins, bathed in whole rat blood, displayed low SCC; skins bathed in 'mammalian' Ringer solution displayed equally low SCC, but responded normally to pitressin or amiloride. 3. When volume expansion was induced in ten rats by infusion of equilibrated whole blood (28 ml/kg body weight) there was a brisk rise in systemic blood pressure, diuresis, natriuresis and kaliuresis. 4. This blood-volume expansion was without detectable effect on the SCC across the skins incorporated within the rats' circulations.