Considerations for the Identification and Conveyance of Clinical Pathology Findings in Preclinical Toxicity Studies: Results From the 9th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology (ESTP) organized a panel of 24 international experts from many fields of toxicologic clinical pathology (e.g., industry, academia, and regulatory) that came together in 2021 to align the use of terminology to convey the importance of clinical pathology findings in preclinical toxicity studies. An additional goal consisted of how to identify important findings in standard and nonstandard clinical pathology associated endpoints. This manuscript summarizes the information and opinions discussed and shared at the ninth ESTP International Expert Workshop, April 5 to 6, 2022. In addition to terminology usage, the workshop considered topics related to the identification and conveyance of the importance of test item-related findings. These topics included sources of variability, comparators, statistics, reporting, correlations to other study data, nonstandard biomarkers, indirect/secondary findings, and an overall weight-of-evidence approach.

Selection and interpretation of clinical pathology indicators of hepatic injury in preclinical studies.

This position paper delineates the expert recommendations of the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology for the use of preclinical, clinical pathology endpoints in assessment of the potential for drug-induced hepatic injury in animals and humans. Development of these guidelines has been based on current recommendations in the relevant preclinical and human clinical trial literature; they are intended to provide a method for consistent and rigorous interpretation of liver-specific data for the identification of hepatic injury in preclinical studies and potential liability for hepatic injury in human patients.

Automated identification of Fos expression.

The concentration of Fos, a protein encoded by the immediate-early gene c-fos, provides a measure of synaptic activity that may not parallel the electrical activity of neurons. Such a measure is important for the difficult problem of identifying dynamic properties of neuronal circuitries activated by a variety of stimuli and behaviours. We employ two-stage statistical pattern recognition to identify cellular nuclei that express Fos in two-dimensional sections of rat forebrain after administration of antipsychotic drugs. In stage one, we distinguish dark-stained candidate nuclei from image background by a thresholding algorithm and record size and shape measurements of these objects. In stage two, we compare performance of linear and quadratic discriminants, nearest-neighbour and artificial neural network classifiers that employ functions of these measurements to label candidate objects as either Fos nuclei, two touching Fos nuclei or irrelevant background material. New images of neighbouring brain tissue serve as test sets to assess generalizability of the best derived classification rule, as determined by lowest cross-validation misclassification rate. Three experts, two internal and one external, compare manual and automated results for accuracy assessment. Analyses of a subset of images on two separate occasions provide quantitative measures of inter- and intra-expert consistency. We conclude that our automated procedure yields results that compare favourably with those of the experts and thus has potential to remove much of the tedium, subjectivity and irreproducibility of current Fos identification methods in digital microscopy.

Polymorphisms of the dopamine D4 receptor and response to antipsychotic drugs.

The dopamine D4 receptor may be a site through which the clinical effects of antipsychotic drugs are mediated. Polymorphisms of a 48 base pair repeat in the third exon of the DRD4 gene code for different length segments in the third intracytoplasmic loop of the D4 receptor. The most common long (seven repeat) form of the D4 receptor has been shown in both physiologic and pharmacologic experiments to respond differently to dopamine agonists and antagonists than do shorter forms of D4. Thus, variants of D4 may partly determine patient response to antipsychotic drugs and, in particular, response to typical neuroleptics, which have a relatively low affinity for the D4 receptor, as compared to clozapine, which has a relatively high affinity for D4. DRD4 polymorphisms in the third intron were characterized in 28 patients with chronic psychosis who responded well to typical neuroleptics, 32 patients who responded well to clozapine, and 57 healthy comparison subjects. Patients responding to typical neuroleptics carried the allele for the long (seven repeat) form of the D4 receptor (allele frequency 8.9%) less frequently than patients responding to clozapine (allele frequency 23.4%, P = 0.046) or healthy comparison subjects (allele frequency 26.3%, P = 0.004). The results of this study suggest that inherited variants of D4 may explain some of the interindividual variation seen in patient response to different classes of antipsychotic medication.

Activation of midline thalamic nuclei by antipsychotic drugs.

The thalamus has been proposed as a site which may be involved in the production of the syndrome of schizophrenia and the response of schizophrenic symptoms to treatment. These studies test whether, consistent with this hypothesis, the activation of thalamic nuclei is a shared property of neuroleptic antipsychotic drugs. Rats were given single doses of the typical high and low potency neuroleptics haloperidol (1 mg/kg) and chlorpromazine (20 mg/kg), the atypical neuroleptics thiroridazine (20 mg/kg) and clozapine (20 mg/kg), the specific dopamine antagonist raclopride (3 mg/kg), the mixed dopamine/serotonin antagonist risperidone (3 mg/kg) or drug-free vehicle. Increased expression of Fos-like protein was utilized as a marker of cellular activation. All drugs tested, including typical and atypical antipsychotic agents, led to similar effects on the midline thalamic paraventricular, centromedian and rhomboid nuclei and the nucleus reuniens. These results suggest that midline thalamic nuclei may participate in neural circuits mediating some of the shared effects of antipsychotic drugs.

Multiplex differential display identifies a novel zinc-finger protein repressed during withdrawal from cocaine.

Multiplex differential display (MDD), a modification of differential display reverse transcriptase polymerase chain reaction (DD-PCR), was used to identify cocaine-dependent regulation of previously known and unknown gene products. Direct comparison of the MDD amplification profiles of duplicate, total RNA samples from the caudate putamen (CPu) of either vehicle or cocaine treated Sprague-Dawley rats indicated that the relative induction of a 240 bp (8G247) product, likely to represent c-fos mRNA, closely paralleled changes in c-fos mRNA as measured by Northern blot analysis. MDD and Northern blot analysis also revealed substantial repression of another PCR product (8G226) at 1 h and 1 day after repeated administration of cocaine. At 2 days after cocaine exposure, the level of 8G226 had returned to control levels. The DNA sequence of 8G226 exhibited near identity with a mouse zinc-finger protein (PZf) and is thus likely to represent a transcriptional regulator. Interestingly, the repression of 8G226 immediately after cocaine treatment is in direct contrast to the cocaine-dependent increase in expression documented for NGFI-A, another zinc-finger protein which also functions as a transcriptional regulator. Detailed characterization of the prolonged reduction in the expression of 8G226 may lead to the identification of additional regulatory pathways that produce changes in cellular response after repeated cocaine exposure.

Mitogenic effects of epidermal growth factor and platelet-derived growth factor on canine and equine mesangial cells in vitro.

OBJECTIVE: To evaluate the effects of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on canine and equine mesangial cell (MC) proliferation in vitro. SAMPLE POPULATION: Third- through eighth-passage canine and equine MC were obtained from explant outgrowth after differential sieving of glomeruli isolated from the kidneys of clinically normal dogs and horses. PROCEDURE: Mitogenic effects of serum, insulin, EGF, and PDGF were evaluated in MC by induction of DNA synthesis, measured as stimulation of [3H]thymidine incorporation and increase in cell numbers. RESULTS: Epidermal growth factor was a potent mitogen in canine and equine MC, stimulating dose-dependent increases in [3H]thymidine incorporation and moderate increase in cell numbers. Although PDGF alone did not significantly stimulate [3H]thymidine incorporation in canine MC, PDGF was synergistic with EGF in stimulation of [3H]thymidine incorporation in canine and equine MC, and PDGF significantly stimulated [3H]thymidine incorporation in equine MC. CONCLUSIONS: Both EGF and PDGF are important mediators of canine and equine MC proliferation. CLINICAL RELEVANCE: Our results are consistent with the hypothesis that growth factors have a role in the progression of glomerular disease in dogs and horses. These findings could prove to be of diagnostic, prognostic, and therapeutic value in the management of spontaneous renal disease in these species.

Acute administration of typical and atypical antipsychotic drugs induces distinctive patterns of Fos expression in the rat forebrain.

Fos expression in the rat brain was investigated by immunohistochemistry after i.p. administration of single doses of a wide range of typical neuroleptic antipsychotic drugs (including the potent dopamine D2 antagonist haloperidol and the mixed monoamine antagonist chlorpromazine) and atypical antipsychotic drugs (including the weak dopamine D2 antagonists clozapine and thioridazine, the relatively pure D2 antagonist raclopride and the mixed D2 and serotonin S2 antagonist risperidone). For comparison to the effects of the antipsychotic drugs and also because the unique clinical therapeutic effects of clozapine have been attributed to S2 blockade, the S2 antagonist ritanserin was also studied. The single shared effect of all antipsychotic drugs tested was the induction of significantly increased Fos immunoreactivity in the nucleus accumbens (NAc). Fos-positive neurons in the NAc were mostly localized in patches throughout its rostrocaudal extent. Haloperidol, chlorpromazine, raclopride and risperidone all significantly increased Fos expression in the medial and lateral striatum. Fos-positive neurons in the striatum were distributed more lateral than medial and declined from rostral to caudal levels. Haloperidol, thioridazine and risperidone also markedly increased Fos expression in the lateral septum. Distinguishing it from the other neuroleptics, clozapine did not increase Fos expression in the lateral striatum, but induced a significant increase in Fos expression in the prefrontal cortex. Ritanserin did not induce Fos expression in any brain region examined, suggesting that S2 antagonism is not responsible for the effects of antipsychotic drugs observed here. Our results suggest that there are distinctive patterns of Fos expression in the forebrain induced by typical and atypical antipsychotic drugs. Notably, Fos expression in the NAc, as a shared property of all the antipsychotic drugs, may be related to the actions mediating the therapeutic effects of these drugs in the treatment of psychotic disorders. The density of Fos-positive neurons stimulated by antipsychotic drugs in the striatum appeared to be correlated with the relative severity of extrapyramidal side-effects produced by these drugs and may be related to the mechanisms mediating these effects.

Persistent reduction of immediate early gene mRNA in rat forebrain following single or multiple doses of cocaine.

Stimulus-induced expression of immediate early genes (IEGs) is believed to be involved in the transduction of extracellular stimuli into prolonged modifications of cellular function. Previous studies have demonstrated that the IEGs NGFI-A (zif268) and c-fos are each rapidly induced in the caudate putamen (CP) by treatment with the indirect dopamine agonist cocaine. The short-term course of this induction has been well studied. However, the consequences of cocaine use are not limited to immediate pharmacological effects. Withdrawal, especially from prolonged or repeated use, can produce extended physiological and behavioral changes. At the cellular level, these longer-term effects may be mediated by or reflected in changes in the expression of IEGs. For this reason, we have investigated long-term perturbations in IEG expression during withdrawal from intravenously (IV) or intraperitoneally (IP) administered cocaine. Levels of NGFI-A and c-fos were measured in the CP of rats by Northern blot analysis, which confirmed that cocaine-induced increases of NGFI-A and c-fos mRNA lasts for several hours after drug administration. Immediately following this induction, however, there is a prolonged period during which a marked reduction in the relative amount of mRNA for both NGFI-A and c-fos is observed in cocaine-treated animals when compared to matched, vehicle-treated controls. This repression persisted for several hours after a single injection and as long as several days following multiple injections, strongly suggesting a cumulative effect for repeated exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.

Two separate 18-amino acid domains of tau promote the polymerization of tubulin.

Tau is a heat-stable microtubule-associated protein which promotes tubulin polymerization. The assembly promoting region of tau was localized using synthetic peptides modeled after domains found in both human and mouse tau. The design of these synthetic peptides was based on the triple repeat motif found in mouse tau. The first peptide, Tau-(187-204), and the second peptide, Tau-(218-235), are capable of promoting the polymerization of tubulin into microtubules, at concentrations above 100 microM. Two other peptides tested, TauR and Tau-(250-267), were not able to promote the assembly of tubulin over a range of concentrations up to 800 microM. TauR is a random analog of Tau-(187-204). Although TauR is unable to promote polymerization, it can modify Tau-(187-204)-induced tubulin assembly.

Induction of the high-affinity nerve growth factor receptor on embryonic chicken sensory nerve cells by elevated potassium.

Culture medium with elevated K+ has been shown to enhance the survival of neurons isolated from several different regions of the nervous system. Nerve growth factor binds to binding sites on sensory and sympathetic neurons through two sites, one of high-affinity (Kd1 approximately 3 X 10(-11) M) and the other of low-affinity (Kd2 approximately 2 X 10(-9) M). Equilibrium binding data generated on dissociated cells derived from E9 chicken embryo dorsal root ganglia, has shown that there is a two-fold increase in the number of high affinity (type I) receptors, with no effect on the affinity, when cells are incubated for 2 hours in buffer containing 59 mM K+. There does not appear to be a significant change in the affinity or the number of low-affinity binding sites. This two-fold increase in type I receptors is dependent on temperature, Ca+2, and active protein synthesis. There does not appear to be an intracellular pool of the type I receptor sufficient to account for this increase. The induction is not observed on sensory nerve cells cultured in 59 mM K+ for 24 hours, either in the presence or absence of nerve growth factor. Additionally, the induction in the number of type I receptors requires that both nerve growth factor and K+ be present simultaneously. Taken in total, this data suggests that there may be a critical period in which the sensory neurons require nerve growth factor exposure to respond. Evidence is presented which indicates that nerve growth factor responsive cells are able to elicit neurites after an acute exposure to nerve growth factor of as little as 4 hours. Finally, there is an approximate two-fold decrease in the concentration of nerve growth factor needed to elicit maximal fiber outgrowth, consistent with the two-fold increase in the number of type I receptors.

Isolation of NILE glycoprotein-related cDNA probes.

The nerve growth factor (NGF)-induced large external (NILE) glycoprotein is an NGF-inducible surface component of PC12 cells that is also widely distributed in the nervous system. It has recently been shown to be indistinguishable from the high-molecular-weight species of the brain antigens L1 and neuron-glia cell adhesion molecule (Ng-CAM) and may have a function in regulating cell adhesion in the developing nervous system. We have used polyclonal anti-NILE antisera to screen a lambda gt11 cDNA library made from NGF-treated PC12 cells. Four molecular probes have been isolated that encode parts of the apoprotein, related proteins, or both. These probes are 1,500, 800, 330, and 300 base pairs long, respectively, and in Northern blots they recognize a family of messages having lengths of approximately 5.9, 3.4, 2.4, and 1.9 kilobases. The two smaller messages are modestly but reproducibly up-regulated by NGF in PC12 cells, as is the NILE glycoprotein; however, only the two larger species would appear to be large enough to encode it. These messages are prominent in brain but not in nonneural tissues, in accordance with the observed levels of the protein. The recombinant phages produce fusion proteins that share specific epitopes with the NILE glycoprotein but not with other proteins. In these experiments, filters coated with recombinant fusion protein were prepared. Antibodies bound to and eluted from these filters specifically immunoprecipitate NILE glycoprotein, but not other proteins, from PC12 cell extracts. Other activities present in the original sera are not specifically retained by recombinant fusion proteins, and Escherichia coli lysates made with nonrecombinant lambda gt11 do not absorb the anti-NILE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor.

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.