Selective membrane toxicity of aminoglycoside antibiotics in membrane vesicles isolated from proximal renal tubules of the rat.

A considerable body of evidence suggests that the nephrotoxic potential of aminoglycoside antibiotics may be associated with the degree of membrane binding and subsequent membrane damage in the renal tubules. In this study, we isolated functional basolateral and luminal membrane vesicles from rat renal cortex, incubated each membrane type in the presence of 1 mM concentrations of either neomycin, netilmicin, gentamicin, hydroxygentamicin, or amikacin, and monitored the activities of the marker enzymes alkaline phosphatase (ALP) and lambda-glutamyltransferase (GGT) (luminal) or ouabain-sensitive Na+,K+-ATPase (basolateral) to determine if there were any selective drug-related alterations of enzyme activities. While none of the five aminoglycosides had any substantive effect upon enzyme activities of luminal vesicles, all five drugs inhibited the basolateral marker enzyme. Neomycin produced the greatest inhibition, hydroxygentamicin and amikacin the least, and gentamicin and netilmicin were intermediate in the inhibition of the enzyme. These results are in accordance with the known relative nephrotoxicity of these same drugs and indicate the usefulness of isolated renal membrane vesicles for in vitro toxicological studies of novel aminoglycosides.

The duration of non-rodent toxicity studies for pharmaceuticals. International Conference on Harmonication (ICH).

At the present time, there are no uniform standards for the duration of non-rodent chronic toxicity studies. The European Union (EU) requires a 6-month non-rodent study. In Japan, a 6-month study is sufficient for most, but not all, compounds. The U.S. Food and Drug Administration (FDA) maintains its standard duration of 12 months for non-rodents, with 6-month studies accepted for some clinical indications on a case-by-case basis. To achieve harmonization on the duration of non-rodent toxicity studies, each member regulatory region (EU, U.S., and Japan) of the International Conference on Harmonization (ICH) collected non-rodent studies with significant new toxicological findings that had occurred after 6 months. An ICH expert working group was organized that included representatives from the regulatory authorities of each ICH region, to jointly review all available case studies for the purpose of arriving at a consensus on the best duration time for non-rodent toxicity studies. Eighteen case studies were identified and evaluated (16 original cases plus 2 additional FDA cases); most of the toxicities identified fell into the following categories: (1) toxicities identified at 6 months; (2) toxicities observed at 12 months, which were absent or considered isolated and not noteworthy findings at 6 months; (3) drug-related deaths or morbidity that occurred between 6 and 12 months, with a pattern of toxicity that permitted the interpolation of findings to an intermediate interval between 6 and 12 months; and (4) a shift in the dose response for toxicity with increasing duration of drug exposure. Of the 18 cases evaluated, 11 supported a study-duration of 9-12 months, 4 supported a duration of 12 months, and the 3 remaining cases indicated that a 6-month study would be adequate. The working group concluded that there was sufficient evidence to support a harmonized 9-month duration for non-rodent toxicity studies, which would be applicable for most categories of pharmaceuticals.

The role of time and size in ontogenetic allometry: I. Review.

Several interconnected issues are a part of most studies of ontogenetic allometry, the relationship between size and shape during growth. One issue is the choice of a model, either linear or one of a series of nonlinear models that have been proposed in the literature. The independent variable, against which growth is assessed, can be a measure of time, e.g., age or a measure of size, e.g., weight. These categories of independent variables have become confused with Medawar's (1945) classification of allometric studies as empirical or deductive. Medawar's distinction may be less useful than Tukey's (1980) between exploratory and confirmatory studies. The "correct" choice for some of these options will be a function of the data set being analyzed. However, an understanding of the implications of these issues is necessary to make the correct choices.

The role of time and size in ontogenetic allometry: II. An empirical study of human growth.

The changes in shape that occur as a consequence of size changes during the third trimester (22 to 36 weeks post conception) of human growth are examined for muscular, tendinous and skeletal measurements of the lower limb. Gestational age and weight were highly correlated, but not linear. Several models appear to have equivalent fits to these data. The skeletal measurements were linear with body weight, but did not follow the predictions of any biomechanical model. Femur length grows more quickly than body weight, as does femur diameter. The ratio of length to diameter is nearly one, suggesting that the relative shape of the femur does not change with weight. The weights of the two muscles examined were nonlinear as functions of age or weight; Gompertz models with different coefficients fits these data best. The lengths of the two tendons were linear with age, but tendon weights were best described by nonlinear Gompertz models. One difficulty of the Gompertz models stems from the lack of an upper asymptote in these data. The data fit the lower half of the curve, however the model predicts an asymptote beyond the range of data available. The relative patterns of growth, i.e. muscles growing faster than tendons, suggest hypotheses that need to be tested with a larger data set. These include growth of muscles relative to bones or tendons in other regions, e.g., the upper limb or the cranium, and an extension of the data in time.

Acetaminophen-induced inhibition of hepatic mitochondrial respiration in mice.

Morphological changes are observed in mitochondria early in the course of acetaminophen (APAP) hepatotoxicity. In order to determine if functional deficits also occur, this study examined the effect of APAP, in vivo and in vitro, on mitochondrial respiration in fasted, male CD-1 mice (3-4 months old). After a hepatotoxic dose of APAP (600 mg/kg, po), when glutamate was used as the respiratory substrate, state 3 respiration (ADP-stimulated) was inhibited and this was reflected in a decreased respiratory control ratio (RCR). In contrast, when succinate was the respiratory substrate, the decreased RCR was reflective of an increase in state 4 (resting) respiration. There was no detectable effect after a nonhepatotoxic dose of APAP (300 mg/kg, po). These APAP-induced respiratory effects and hepatotoxicity were prevented by piperonyl butoxide pretreatment, and were absent in 1- and 2-month-old mice, which are resistant to APAP-induced damage. Since the APAP-induced inhibition of mitochondrial respiration, in vivo, correlated with age-related and piperonyl butoxide-dependent differences in toxicity, the data suggest that the in vivo effects result, at least in part, from a mixed-function oxidase generated metabolite. In vitro, both state 3 and state 4 respiration, as well as the RCR, were inhibited by APAP in a concentration-dependent manner with glutamate as substrate. However, no effects were observed with succinate as substrate, thereby contrasting with results obtained following in vivo exposure. Therefore the in vitro effects of APAP are different from those observed in vivo and may result from a direct insult of the parent compound. These studies suggest that early alterations in mitochondrial function may be mechanistically important in APAP hepatotoxicity.

Mechanical instability as a cause of gait disturbance in high-grade spondylolisthesis: a pre- and postoperative three-dimensional gait analysis.

Mechanical instability of the spinopelvic junction is a suspected cause of abnormal gait in high-grade spondylolisthesis. Computerized three-dimensional gait analysis was performed on a 10-year-old with grade III spondylolisthesis at L-5. Preoperatively, the gait pattern was characterized by posterior pelvic tilt, decreased hip flexion, increased knee flexion, and decreased stride length and walking speed. All temporal and kinematic parameters of gait normalized after laminectomy and instrumented, in situ arthrodesis (L-4-S-1). The absence of any neurologic abnormalities on preoperative imaging, intraoperative somatosensory-evoked potentials (SSEP) monitoring, and nerve-root exploration, together with the observed improvement after stabilization of the spinopelvic junction, suggests a mechanical basis for the gait changes in high-grade spondylolisthesis.