Fundamental and Applied Toxicology最新文献

Seasonal hyporesponsiveness and other immune system variations were observed in female B6C3F1 mice during routine screening tests for immunomodulation. In a retrospective assessment, 4 years of data from over 1200 naive, vehicle, and immunosuppressed (cyclophosphamide-treated) control mice were compiled and analyzed for uniformity and significant circannual pattern of immune response. Endpoints included body, spleen, and thymus weights and an immunotoxicity assessment which enumerates specific antibody plaque-forming cells (PFC) in the spleen following immunization with sheep red blood cells. Dosing vehicles were water, corn oil, or 1% methyl cellulose instilled by oral gavage in a 5–20 ml/kg volume once daily for 5 days. Four days later, terminal organ and body weights were recorded and PFC were quantitated. Upon analysis, individual datapoints were arrayed in consistent circannual and seasonal patterns. In naive mice, the yearly peak response in circannual rhythm (acrophase) for body weight and PFC parameters occurred in the summer, with acrophases for spleen and thymus weights located in the spring. Vehicle gavage modulated the circannual/seasonal means and acrophases of all measured endpoints in distinct patterns which varied by vehicle. Body weight was the endpoint least affected by vehicle treatment. Corn oil was the vehicle resulting in the most dramatic effects on natural rhythm. As expected, the naive mice receiving an ip injection of cyclophosphamide exhibited significant decreases (p≤ 0.05) in circannual mean values for PFC response and relative organ weights when compared to naive controls and the elimination of significant expression of rhythm for PFC parameters. Our results indicate that dosing vehicles alter normal seasonal patterns of biological responses in the mouse. These effects on natural rhythms should be considered in toxicity evaluations, especially when comparing datapoints collected at different times of the year.

It is difficult to understand why culling (reduction of litter size) has become such a widely used procedure in reproductive toxicity studies since there appear to have been no prior investigations to ascertain that it would improve the efficiency of studies with respect to detecting adverse effects. Perhaps the only provable advantage of culling is with respect to economics and convenience.Post hocrationalizations for culling lack conviction because many of the claims made for culling are erroneous, inconsistent, vague, and contradictory. Mostly, they are based on part truths derived from minimal studies, conducted for totally different purposes. That experimental animals have to be killed sooner or later is unquestioned, but for ethical and scientific reasons, it is imperative that the maximum amount of information is obtained from them. Currently, the most common practice is to cull litters to four per sex (total eight) on Day 4 postpartum. This is totally divorced from natural values for most rat strains and involves elimination, usually without adequate examination, of between 30 and 45% of offspring. Without culling most of these would survive, unless there was a treatment effect. Intuitively, it would seem that removal of such a proportion of offspring would severely limit the possibility of detecting the postnatal equivalent of fetal malformations. Culling totally nullifies litter size as an indicator of toxicity. Indirectly, it also nullifies the value of mean pup weight as an indicator of toxicity because it greatly increases the variation in mean pup weight. This is quite contrary to the claim that culling reduces variance. Further, the increased growth of offspring in culled litters can have long-term consequences of a shorter overall and reproductive life span.

The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 μg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised (≈30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP.

The differential inhibition of the target esterases acetylcholinesterase (AChE) and neuropathy target esterase (NTE, neurotoxic esterase) by organophosphorus compounds (OPs) is followed by distinct neurological consequences in exposed subjects. The present study demonstrates that neuroblastoma cell lines (human SH-SY5Y and murine NB41A3) can be used to differentiate between neuropathic OPs (i.e., those inhibiting NTE and causing organophosphorus-induced delayed neuropathy) and acutely neurotoxic OPs (i.e., those highly capable of inhibiting AChE). In these experiments, concentration–response data indicated that the capability to inhibit AChE was over 100× greater than the capability to inhibit NTE for acutely toxic, nonneuropathic OPs (e.g., paraoxon and malaoxon) in both cell lines. Inhibition of AChE was greater than inhibition of NTE, without overlap of the concentration–response curves, for OPs which are more likely to cause acute, rather than delayed, neurotoxic effectsin vivo(e.g., chlorpyrifos-oxon, dichlorvos, and trichlorfon). In contrast, concentrations inhibiting AChE and NTE overlapped for neuropathy-causing OPs. For example, apparent IC50 values for NTE inhibition were less than 9.6-fold the apparent IC50 values for AChE inhibition when cells were exposed to the neuropathy-inducing OPs diisopropyl phosphorofluoridate, cyclic tolyl saligenin phosphate, phenyl saligenin phosphate, mipafox, dibutyl dichlorovinyl phosphate, and di-octyl-dichlorovinyl phosphate. In all cases, esterase inhibition occurred at lower concentrations than those needed for cytoxicity. These results suggest that either mouse or human neuroblastoma cell lines can be considered usefulin vitromodels to distinguish esterase-inhibiting OP neurotoxicants.

Based on a review of the pertinent literature and our own unpublished data, it is recommended that culling of rodent litters in the early postnatal period should be a standard practice in delivery-type reproduction studies. This, in turn, will reduce the litter size-induced variability in the growth and development of pups during the postnatal period and thus increase the sensitivity of statistical analyses to detect treatment-related effects. This will also ensure that any adverse effects on pup growth (body weight gain) and development (reflex and behavior development) are not masked by a treatment-induced reduction in litter size. The culling should be carried out randomly and no attempt should be made to selectively cull sick or underweight pups. Since male pups weigh significantly more than females and studies have shown differences in maternal behavior toward one sex over the other, whenever possible each culled litter should consist of an equal number of males and females.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca²⁺-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr⁵¹release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Male CD- 1 mice were fed diets containing 0 (control), 10, 30, 100, and 300 mg/kg/day piperonyl butoxide (PBO) and 0.05% sodium phenobarbital (NaPB) and male F344 rats were fed diets containing 0 (control), 100, 550, 1050, and 1850 mg/kg/day PBO and 0.5% NaPB for periods of 7 and 42 days. In both species PBO and NaPB increased relative liver weight and whereas PBO produced a midzonal (mouse) or periportal/midzonal (rat) hypertrophy, NaPB produced a centrilobular hypertrophy. In the rat, individual cell necrosis was also observed at 42 days after high doses of PBO. Replicative DNA synthesis, assessed as the hepatocyte labeling index following implantation of 7-day osmotic pumps containing 5-bromo-2′-deoxyuridine during Study Days 0–7 and 35-42, was increased in mice given 300 mg/kg/day PBO and NaPB for 7 days and in rats given 550 and 1050 mg/kg/day PBO and NaPB for 7 days and 1050 mg/kg/day PBO for 42 days. While PBO had no effect on body weights in mice, the body weights of rats given 550, 1050, and 1850 mg/kg/day PBO for 42 days were reduced to 92, 89, and 70% of control, respectively. PBO induced microsomal cytochrome P450 content and mixed function oxidase activities in the mouse and rat, although the effects were less marked than those produced by NaPB. In summary, this data demonstrates that PBO can produce liver enlargement in the mouse and the rat which is associated with induction of xenobiotic metabolism, hypertrophy, and hyperplasia. The hepatic effects of PBO in the mouse were similar to but less marked than those produced by NaPB. In the rat high doses of PBO were hepatotoxic and resulted in a marked reduction in body weight. Thus while the reported formation of eosinophilic nodules in mouse liver by PBO may occur by a mechanism(s) similar to that of NaPB and other nongenotoxic enzyme inducers, the reported tumor formation in rats at greater than the maximum tolerated dose is most likely associated with marked enzyme induction in conjunction with a regenerative hyperplasia resulting from PBO-induced hepatotoxicity.

Mitochondria have long been known to participate in the process of cell injury associated with metabolic failure. Only recently, however, have we come to appreciate the role of mitochondria as primary intracellular targets in the initiation of cell dysfunction. In addition to ATP synthesis, mitochondria are also critical to modulation of cell redox status, osmotic regulation, pH control, and cytosolic calcium homeostasis and cell signaling. Mitochondria are susceptible to damage by oxidants, electrophiles, and lipophilic cations and weak acids. Chemical-induced mitochondrial dysfunction may be manifested as diverse bioenergetic disorders and considerable effort is required to distinguish between mechanisms involving critical mitochondrial targets and those in which mitochondrial dysfunction is secondary and plays only a modulatory role in cell injury. The following paragraphs review a few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial metabolism and bioenergetics, gene regulation, or signal transduction in the form of apoptosis and altered cell cycle control. Greater understanding of the molecular mechanisms of mitochondrial bioenergetics, ion regulation, and genetics will lead to numerous additional examples of mitochondria-mediated cell injury, revealing important new insight regarding the prediction, prevention, diagnosis, and treatment of chemical-induced toxic tissue injury.

To define the kinetics and safety of spinally infused recombinant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF), beagle dogs were prepared with lumbar intrathecal catheters passed through the cisternal membrane to the L1–L4 lumbar level. For kinetic studies, r-metHuBDNF was delivered by bolus or infusion through one catheter and lumbar CSF was sampled periodically through a second. As a lumbar bolus, r-metHuBDNF displayed a biphasic clearance witht$\text{1}\text{2}$a = 0.7 hr andt$\text{1}\text{2}$b = 7.9 hr. Lumbar to cisternal concentrations after bolus delivery were approximately 60:1. For safety studies, dogs received continuous intrathecal infusion (2.4 ml/day) for 28 days of saline (n= 6), r-metHuBDNF at 200 (n= 6), 800 (n= 6), or 2000 (n= 7) μg/day. Control dogs showed no changes. Intrathecally infused r-metHuBDNF produced a dose-dependent increase in muscle tone and decreased coordination. Low-dose r-metHuBDNF was associated with moderate increases in muscle tone after 22–28 days of infusion. No clinically important changes were noted in rectal temperature, arterial pressure, respiration and heart rate, body weight, food consumption, stool or urine output, or change in blood chemistries measured throughout the study. Cisternal CSF protein and glucose sampled at 28 days were not different between dose groups and all cultures were negative. Histopathological examination of the spinal cord typically revealed some degree of chronic inflammation around the catheter, including fibrotic adhesions and focal accumulations of lymphoid and plasma cells, but these effects were not dose dependent. In other dogs receiving r-metHuBDNF (2000 or 4000 μg/day), termination of infusion resulted in significant recovery.